Antibodies against human CSF-1R and uses thereof

ABSTRACT

The present invention relates to antibodies against human CSF-1R (anti-CSF-1R antibody), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/640,183, filed Mar. 6, 2015, which is a Divisional of U.S. patentapplication Ser. No. 12/962,445, filed Dec. 7, 2010, now U.S. Pat. No.8,999,327, issued Apr. 7, 2015, which claims the benefit of EP PatentApplication Nos. 09 015 310.7, filed Dec. 10, 2009, and 10 173 407.7,filed Aug. 19, 2010, the entire disclosures of which are expresslyincorporated by reference herein.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392029612SEQLIST.txt,date recorded: Oct. 12, 2016, size: 68 KB).

FIELD OF THE INVENTION

The present invention relates to antibodies against human CSF-1R(anti-CSF-1R antibodies), methods for their production, pharmaceuticalcompositions containing said antibodies, and uses thereof.

BACKGROUND OF THE INVENTION

The human CSF-1 receptor (CSF-1R; colony stimulating factor 1 receptor;synonyms: M-CSF receptor; Macrophage colony-stimulating factor 1receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 62) is known since 1986(Coussens, L., et al., Nature 320 (1986) 277-280). CSF-1R is a growthfactor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth, Pand Stanley, E. R., Curr. Top. Microbiol Immunol. 181 (1992) 141-67).

CSF-1R is the receptor for CSF-1 colony stimulating factor 1, alsocalled M-CSF, macrophage colony-stimulating factor) and mediates thebiological effects of this cytokine (Sherr, C. J., et al., Cell 41(1985) 665-676). The cloning of the colony stimulating factor-1 receptor(CSF-1R) (also called c-fms) was described for the first time inRoussel, M. F., et al., Nature 325 (1987) 549-552. In that publication,it was shown that CSF-1R had transforming potential dependent on changesin the Cterminal tail of the protein including the loss of theinhibitory tyrosine 969 phosphorylation which binds Cbl and therebyregulates receptor down regulation (Lee, P. S., et al., Embo J. 18(1999) 3616-3628). Recently a second ligand for CSF-1R termedinterleukin-34 (IL-34) was identified (Lin, H. et al, Science 320 (2008)807-811).

The cytokine CSF-1 (colony stimulating factor 1, also called M-CSF,macrophage) is found extracellularly as a disulfide-linked homodimer(Stanley, E. R. et al., Journal of Cellular Biochemistry 21 (1983)151-159; Stanley, E. R. et al., Stem Cells 12 Suppl. 1 (1995) 15-24).

The main biological effects of CSF-1R signaling are the differentiation,proliferation, migration, and survival of hematopoietic precursor cellsto the macrophage lineage (including osteoclast). Activation of CSF-1Ris mediated by its ligands, CSF-1 (M-CSF) and IL-34. Binding of CSF-1(M-CSF) to CSF-1R induces the formation of homodimers and activation ofthe kinase by tyrosine phosphorylation (Li, W. et al, EMBO Journal.10(1991) 277-288; Stanley, E. R., et al., Mol. Reprod. Dev. 46 (1997)4-10).

The biologically active homodimer CSF-1 binds to the CSF-1R within thesubdomains D1 to D3 of the extracellular domain of the CSF-1 receptor(CSF-1R-ECD). The CSF-1R-ECD comprises five immunoglobulin-likesubdomains (designated D1 to D5). The subdomains D4 to D5 of theextracellular domain (CSF-1R-ECD)are not involved in the CSF-1 binding.(Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359).The subdomain D4 is involved in dimerization (Yeung, Y-G., et alMolecular & Cellular Proteomics 2 (2003) 1143-1155; Pixley, F. J., etal., Trends Cell Biol 14 (2004) 628-638).

Further signaling is mediated by the p85 subunit of PI3K and Grb2connecting to the PI3K/AKT and Ras/MAPK pathways, respectively. Thesetwo important signaling pathways can regulate proliferation, survivaland apoptosis. Other signaling molecules that bind the phosphorylatedintracellular domain of CSF-1R include STAT1, STAT3, PLCy, and Cbl(Bourette, R. P. and Rohrschneider, L. R., Growth Factors 17 (2000)155-166).

CSF-1R signaling has a physiological role in immune responses, in boneremodeling and in the reproductive system. The knockout animals foreither CSF-1 (Pollard, J. W., Mol. Reprod. Dev. 46 (1997) 54-61) orCSF-1R (Dai, X. M., et al., Blood 99 (2002) 111-120) have been shown tohave osteopetrotic, hematopoietic, tissue macrophage, and reproductivephenotypes consistent with a role for CSF-1R in the respective celltypes.

Sherr, C. J, et al., Blood 73 (1989) 1786-1793 relates to someantibodies against CSF-1R that inhibit the CSF-1 activity (see Sherr, C.J. et al., Blood 73 (1989) 1786-1793). Ashmun, R. A., et al., Blood 73(1989) 827-837 relates to CSF-1R antibodies. Lenda, D., et al., Journalof Immunology 170 (2003) 3254-3262 relates to reduced macrophagerecruitment, proliferation, and activation in CSF-1-deficient miceresults in decreased tubular apoptosis during renal inflammation.Kitaura, H., et al., Journal of Dental Research 87 (2008) 396-400 refersto an anti-CSF-1 antibody which inhibits orthodontic tooth movement, WO2001/030381 mentions CSF-1 activity inhibitors including antisensenucleotides and antibodies while disclosing only CSF-1 antisensenucleotides. WO 2004/045532 relates to metastases and bone lossprevention and treatment of metastatic cancer by a CSF-1 antagonistdisclosing as antagonist anti-CSF-1-antibodies only. WO 2005/046657relates to the treatment of inflammatory bowel disease byanti-CSF-1-antibodies. US 2002/0141994 relates to inhibitors of colonystimulating factors. WO 2006/096489 relates to the treatment ofrheumatoid arthritis by anti-CSF-1-antibodies. WO 2009/026303 and WO2009/112245 relate to certain anti-CSF-1R antibodies binding to CSF-1Rwithin the first three subdomains (D1 to D3) of the Extracellular Domain(CSF-1R-ECD).

SUMMARY OF THE INVENTION

The invention comprises an isolated antibody binding to human CSF-1R,wherein the antibody binds to human CSF-1R fragment delD4 (SEQ ID NO:65) and to human CSF-1R Extracellular Domain (SEQ ID NO: 64) with aratio of 1:50 or lower.

The invention further comprises an isolated antibody wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ ID NO:15 and        the light chain variable domain comprises SEQ ID NO:16;    -   c) the heavy chain variable domain comprises SEQ ID NO:75 and        the light chain variable domain comprises SEQ NO:76;    -   d) the heavy chain variable domain comprises SEQ ID NO:83 and        the light chain variable domain comprises SEQ ID NO:84;

or a humanized version thereof.

The invention further comprises an isolated antibody wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ NO:15 and the        light chain variable domain comprises SEQ ID NO: 16;

or a humanized version thereof.

In one embodiment the isolated antibody comprises

-   -   a) a heavy chain variable domain comprising SEQ ID NO:23 and a        light chain variable domain comprising SEQ ID NO:24, or    -   b) a heavy chain variable domain comprising SEQ ID NO:31 and a        light chain variable domain comprising SEQ ID NO:32, or    -   c) a heavy chain variable domain comprising SEQ ID NO:39 and a        light chain variable domain comprising SEQ NO:40, or    -   d) a heavy chain variable domain comprising SEQ ID NO:47 and a        light chain variable domain comprising SEQ ID NO:48, or    -   e) a heavy chain variable domain comprising SEQ ID NO:55 and a        light chain variable domain comprising SEQ ID NO:56.

The invention further comprises an isolated antibody, wherein

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 1, a CDR2 region comprising SEQ ID NO: 2,        and a CDR1 region comprising SEQ ID NO:3, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO: 4,        a CDR2 region comprising SEQ ID NO:5, and a CDR1 region        comprising SEQ NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 9, a CDR2 region comprising SEQ ID NO: 10,        and a CDR1 region comprising SEQ ID NO: 11, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:12,        a CDR2 region comprising SEQ ID NO: 13, and a CDR1 region        comprising SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ IT) NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ NO: 51, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:52,        a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54; or    -   h) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:69, a CDR2 region comprising SEQ ID NO: 70,        and a CDR1 region comprising SEQ ID NO:71, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        72, a CDR2 region comprising SEQ ID NO:73, and a CDR1 region        comprising SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 77, a CDR2 region comprising SEQ ID NO:        78, and a CDR1 region comprising SEQ ID NO: 79, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:80, a CDR2 region comprising SEQ ID NO: 81, and a CDR1 region        comprising SEQ ID NO: 82.

Preferably the antibody according to the invention is of human IgG1subclass or of human IgG4 subclass.

A further embodiment of the invention is a pharmaceutical compositioncomprising any of the antibodies described herein.

The invention further comprises the use an of an antibody according tothe invention for the manufacture of a medicament for treatment of aCSF-1R mediated disease.

The invention further comprises the use an of an antibody according tothe invention for the manufacture of a medicament for treatment ofcancer.

The invention further comprises the use an of an antibody according tothe invention for the manufacture of a medicament for treatment of boneloss.

The invention further comprises the use an of an antibody according tothe invention for the manufacture of a medicament for treatment ofmetastasis.

The invention further comprises the use an of an antibody according tothe invention for the manufacture of a medicament for treatment ofinflammatory diseases.

The invention further comprises an antibody according to the inventionfor treatment of a CSF-1R mediated disease.

The invention further comprises an antibody according to the inventionfor treatment of cancer.

The invention further comprises an antibody according to the inventionfor treatment of bone loss.

The invention further comprises an antibody according to the inventionfor treatment of metastasis.

The invention further comprises an antibody according to the inventionfor treatment of inflammatory diseases.

A further embodiment of the invention is a nucleic acid encoding anantibody wherein.

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 1, a CDR2 region comprising SEQ ID NO: 2,        and a CDR1 region comprising SEQ ID NO:3, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO: 4,        a CDR2 region comprising SEQ ID NO:5, and a CDR1 region        comprising SEQ NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 9, a CDR2 region comprising SEQ ID NO: 10,        and a CDR1 region comprising SEQ ID NO: 11, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:12,        a CDR2 region comprising SEQ ID NO: 13, and a CDR1 region        comprising SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, of    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ ID NO: 51, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:52, a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54, or    -   h) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:69, a CDR2 region comprising SEQ ID NO: 70,        and a CDR1 region comprising SEQ ID NO:71, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        72, a CDR2 region comprising SEQ ID NO:73, and a CDR1 region        comprising SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 77, a CDR2 region comprising SEQ ID NO:        78, and a CDR1 region comprising SEQ ID NO: 79, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:80, a CDR2 region comprising SEQ ID NO: 81, and a CDR1 region        comprising SEQ ID NO: 82,

A further embodiment of the invention is a nucleic acid encoding anantibody wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ ID NO:15 and        the light chain variable domain comprises SEQ ID NO:16;    -   c) the heavy chain variable domain comprises SEQ ID NO:75 and        the light chain variable domain comprises SEQ ID NO:76;    -   d) the heavy chain variable domain comprises SEQ ID NO:83 and        the light chain variable domain comprises SEQ ID NO:84;        or a humanized version thereof.

A further embodiment of the invention is a nucleic acid encoding anantibody wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:23 and        the light chain variable domain comprises SEQ ID NO:24, or    -   b) the heavy chain variable domain comprises SEQ ID NO:31 and        the light chain variable domain comprises SEQ ID NO:32, or    -   c) the heavy chain variable domain comprises SEQ ID NO:39 and        the light chain variable domain comprises SEQ ID NO:40, or    -   d) the heavy chain variable domain comprises SEQ ID NO:47 and        the light chain variable domain comprises SEQ ID NO:48, or    -   e) the heavy chain variable domain comprises SEQ ID NO:55 and        the light chain variable domain comprises SEQ ID NO:56.

The invention further provides expression vectors containing nucleicacid according to the invention capable of expressing said nucleic acidin a prokaryotic or eukaryotic host cell, and host cells containing suchvectors for the recombinant production of an antibody according to theinvention.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention.

The invention further comprises a method for the production of arecombinant human or humanized antibody according to the invention, themethod comprising expressing a nucleic acid according to the inventionin a prokaryotic or eukaryotic host cell and recovering said antibodyfrom said cell or the cell culture supernatant. The invention furthercomprises the antibody obtained by such a recombinant method.

Antibodies according to the invention show benefits for patients in needof a CSF-1R targeting therapy. The antibodies according to the inventionshow efficient antiproliferative activity against ligand-independent andligand-dependant proliferation and are therefore especially useful inthe treatment of cancer and metastasis.

The invention further provides a method for treating a patient sufferingfrom cancer, the method comprising administering to a patient diagnosedas having such a disease (and therefore being in need of such a therapy)an effective amount of an antibody according to the invention. Theantibody is administered preferably in a pharmaceutical composition.

A further embodiment of the invention is a method for treating a patientsuffering from cancer the method comprising administering to the patientan antibody according to the invention.

These and other embodiments of the invention are further described inthe detailed description that follows.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 heavy chain CDR3, Mab 2F11

SEQ ID NO: 2 heavy chain CDR2, Mab 2F11

SEQ ID NO: 3 heavy chain CDR1, Mab 2F11

SEQ ID NO: 4 light chain CDR3, Mab 2F11

SEQ ID NO: 5 light chain CDR2, Mab 2F11

SEQ ID NO: 6 light chain CDR1, Mab 2F11

SEQ ID NO: 7 heavy chain variable domain, Mab 2E11

SEQ ID NO: 8 light chain variable domain, Mab 2E11

SEQ ID NO: 9 heavy chain CDR3, Mab 2E10

SEQ ID NO: 10 heavy chain CDR2, Mab 2E10

SEQ ID NO: 11 heavy chain CDR1, Mab 2E10

SEQ ID NO: 12 light chain CDR3, Mab 2E10

SEQ ID NO: 13 light chain CDR2, Mab 2E10

SEQ ID NO: 14 light chain CDR1, Mab 2E10

SEQ ID NO: 15 heavy chain variable domain, Mab 2E10

SEQ ID NO: 16 light chain variable domain, Mab 2E10

SEQ ID NO: 17 heavy chain CDR3, hMab 2F11-c11

SEQ ID NO: 18 heavy chain CDR2, hMab 2F11-c11

SEQ ID NO: 19 heavy chain CDR1, hMab 2F11-c11

SEQ ID NO: 20 light chain CDR3, hMab 2F11-c11

SEQ ID NO: 21 light chain CDR2, hMab 2F11-c11

SEQ ID NO: 22 light chain CDR1, hMab 2F11-c11

SEQ ID NO: 23 heavy chain variable domain, hMab 2F11-c11

SEQ ID NO: 24 light chain variable domain, hMab 2F11-c11

SEQ ID NO: 25 heavy chain CDR3, hMab 2F11-d8

SEQ ID NO: 26 heavy chain CDR2, hMab 2F11-d8

SEQ ID NO: 27 heavy chain CDR1, hMab 2F11-d8

SEQ ID NO: 28 light chain CDR3, hMab 2F11-d8

SEQ ID NO: 29 light chain CDR2, hMab 2F11-d8

SEQ ID NO: 30 light chain CDR1, hMab 2F11-d8

SEQ ID NO: 31 heavy chain variable domain, hMab 2F11-d8

SEQ ID NO: 32 light chain variable domain, hMab 2F11-d8

SEQ ID NO: 33 heavy chain CDR3, hMab 2F11-e7

SEQ ID NO: 34 heavy chain CDR2, hMab 2F11-e7

SEQ ID NO: 35 heavy chain CDR1, hMab 2F11-e7

SEQ ID NO: 36 light chain CDR3, hMab 2F11-e7

SEQ ID NO: 37 light chain CDR2, hMab 2F11-e7

SEQ ID NO: 38 light chain CDR1, hMab 2F11-e7

SEQ ID NO: 39 heavy chain variable domain, hMab 2F11-e7

SEQ ID NO: 40 light chain variable domain, hMab 2F11-e7

SEQ ID NO: 41 heavy chain CDR3, hMab 2F11-f12

SEQ ID NO: 42 heavy chain CDR2, hMab 2F11-f12

SEQ ID NO: 43 heavy chain CDR1, hMab 2F11-f12

SEQ ID NO: 44, light chain CDR3, hMab 2F11-f12

SEQ ID NO: 45 light chain CDR2, hMab 2F11-f12

SEQ ID NO: 46 light chain CDR1, hMab 2F11-f12

SEQ ID NO: 47 heavy chain variable domain, hMab 2F11-f12

SEQ ID NO: 48 light chain variable domain, hMab 2F11-f12

SEQ ID NO: 49 heavy chain CDR3, hMab 2F11-g1

SEQ ID NO: 50 heavy chain CDR2, hMab 2F11-g1

SEQ ID NO: 51 heavy chain CDR1, hMab 2F11-g1

SEQ ID NO: 52 light chain CDR3, hMab 2F11-g1

SEQ ID NO: 53 light chain CDR2, hMab 2F11-g1

SEQ ID NO: 54 light chain CDR1, hMab 2F11-g1

SEQ ID NO: 55 heavy chain variable domain, hMab 2F11-g1

SEQ ID NO: 56 light chain variable domain, hMab 2F11-g1

SEQ ID NO: 57 human kappa light chain constant region

SEQ ID NO: 58 human heavy chain constant region derived from IgG1

SEQ ID NO: 59 human heavy chain constant region derived from IgG1mutated on L234A and L235A

SEQ ID NO: 60 human heavy chain constant region derived from IgG4

SEQ ID NO: 61 human heavy chain constant region derived from IgG4mutated on S228P

SEQ ID NO: 62 human wildtype CSF-1R (wt CSF-1R)

SEQ ID NO: 63 human mutant CSF-1R L3O1S Y969F

SEQ ID NO: 64 human CSF-1R Extracellular Domain

SEQ ID NO: 65 human CSF-1R fragment delD4

SEQ ID NO: 66 human CSF-1R fragment D1-D3

SEQ ID NO: 67 signal peptide

SEQ ID NO: 68 Primer

SEQ ID NO: 69 heavy chain CDR3, Mab 1G10

SEQ ID NO: 70 heavy chain CDR2, Mab 1G10

SEQ ID NO: 71 heavy chain CDR1, Mab 1G10

SEQ ID NO: 72 light chain CDR3, Mab 1G10

SEQ ID NO: 73 light chain CDR2, Mab 1G10

SEQ ID NO: 74 light chain CDR1, Mab 1G10

SEQ ID NO: 75 heavy chain variable domain, Mab 1G10

SEQ ID NO: 76 light chain variable domain, Mab 1G10

SEQ ID NO: 77 heavy chain CDR3, Mab 2H7

SEQ ID NO: 78 heavy chain CDR2, Mab 2H7

SEQ ID NO: 79 heavy chain CDR 1, Mab 2H7

SEQ ID NO: 80 light chain CDR3, Mab 2H7

SEQ ID NO: 81 light chain CDR2, Mab 2H7

SEQ ID NO: 82 light chain CDR1, Mab 2H7

SEQ ID NO: 83 heavy chain variable domain, Mab 2H7

SEQ ID NO: 84 light chain variable domain, Mab 2H7

DESCRIPTION OF THE FIGURES

FIG. 1A-1B Growth inhibition of BeWo tumor cells in 3D culture undertreatment with different anti-CSF-1R monoclonal antibodies at aconcentration of 10 μg/ml. X axis: viability normalized mean relativelight units (RLU) corresponding to the ATP-content of the cells(CellTiterGlo assay). Y axis: tested probes: Minimal Medium (0.5% FBS),mouse IgG1 (mIgG1, 10 μg/ml), mouse IgG2a (mIgG2a 10 μg/ml), CSF-1 only,Mab 2F11, Mab 2E10, Mab2H7, Mab 1G10 and SC 2-4A5. Highest inhibition ofCSF-1 induced growth was observed with the anti-CSF-1R antibodiesaccording to the invention.

FIG. 2A Biacore sensogram of binding of different anti-CSF-1R antibodiesto immobilized human CSF-1R fragment delD4 (comprising the extracellularsubdomains D1-D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal inResponse Units (RU), baseline=0 RU, x-axis: time in seconds(s)): Whilethe antibodies Mab 3291 and sc 2-4A5 clearly show binding to this delD4fragment, the antibodies according to the invention e.g. Mab 2F11, andMab 2E10, did not bind to the CSF-1R fragment delD4. The controlanti-CCR5 antibody m<CCR5>Pz03.1C5 did also not bind to the CSF-1Rfragment delD4.

FIG. 2B Biacore sensogram of binding of different anti-CSF-1R antibodiesto immobilized human CSF-1R Extracellular Domain (CSF-1R-ECD)(comprising the extracellular subdomains D1-D5) (SEQ ID NO: 64) (y-axis:binding signal in Response Units (RU), baseline=0 RU, x-axis: time inseconds(s)):: All anti-CSF-1R antibodies show binding to CSF-1R-ECD. Thecontrol anti-CCR5 antibody m<CCR5>Pz03.1C5 did not bind, to theCSF-1R-ECD.

FIG. 2C Biacore sensogram of binding of different anti-CSF-1R antibodiesto immobilized human CSF-1R fragment delD4 (comprising the extracellularsubdomains D1-D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal inResponse Units (RU), baseline=0 RU, x-axis: time in seconds (s)): Mab1G10, Mab 2H7 and humanized hMab 2F11-e7 did not bind to the CSF-1Rfragment delD4. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did alsonot bind to the CSF-1R fragment delD4.

FIG. 2D Biacore sensogram of binding of different anti-CSF-1R antibodiesto immobilized human CSF-1R Extracellular Domain (CSF-1R-ECD)(comprising the extracellular subdomains D1-D5) (SEQ ID NO: 64) (y-axis:binding signal in Response Units (RU), baseline=0 RU, x-axis: time inseconds (s)): All anti-CSF-1R antibodies Mab 1G10, Mab 2H17 andhumanized hMab 2F11-e7 showed binding to CSF-1R-ECD. The controlanti-CCR5 antibody m<CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD.

FIG. 2E Biacore sensogram of binding of different anti-CSF-1R antibodiesto immobilized human CSF-1R fragment delD4 (comprising the extracellularsubdomains D1-D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal inResponse Units (RU), baseline=0 RU, x-axis: time in seconds(s)): Allanti-CSF-1R antibodies 1.2.SM, CXIIG6, ab10676 and MAB3291 show bindingto to the CSF-1R fragment delD4. The control anti-CCR5 antibodym<CCR5>Pz03.1C5 did also not bind to the CSF-1R fragment delD4.

FIG. 2F Biacore sensogram of binding of different anti-CSF-1R antibodiesto immobilized human CSF-1R Extracellular Domain (CSF-1R-ECD)(comprising the extracellular subdomains D1-D5) (SEQ ID NO: 64) (y-axis:binding signal in Response Units (RU), baseline=0 RU, x-axis: time inseconds(s)):: All anti-CSF-1R antibodies 1.2.SM, CXIIG6, ab10676 andMAB3291 show binding to CSF-1R-ECD. The control anti-CCR5 antibodym<CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD.

FIG. 3A-3D CSF-1 levels in Cynomolgus monkey after application ofdifferent dosages of anti-CSF-1R antibody according to the invention

FIG. 4 In vivo efficacy—tumor growth inhibition of anti-CSF-1Rantibodies according to the invention in breast cancer BT20 xenograft

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides compositions that bind human CSF-1R or aportion thereof, kits and articles of manufacture comprising suchcompositions, and methods of using such compositions, including, e.g.,methods for modulating ligand binding to CSF-1R and for modulatingbiological/physiological activities associated with ligand binding toCSF-1R. The invention is based in part on the identification of avariety of anti-CSF-1R antibodies that bind CSF-1R. The anti-CSF-1Rantibodies of the invention can conveniently be used as therapeutic anddiagnostic agents for use in targeting CSF-1R-mediated diseasesincluding, e.g., cancer (including, for example, metastatic cancer),bone disorders (including, for example, bone loss), and inflammatorydisorders.

Surprisingly it has been found that, using a human CSF-1R fragment delD4in which the D4 subdomain of human CSF-1R-ECD was deleted (SEQ IDNO:65), the new anti-CSF-1R antibodies according to the invention couldbe selected. These antibodies show valuable properties like excellentligand-dependant cell growth inhibition and at the same time ligandindependent cell growth inhibition of NIH 3T3 cell, retrovirallyinfected with either an expression vector for full-length wildtypeCSF-1R (SEQ ID NO:62) or mutant CSF-1R L301S Y969F (SEQ ID NO:63)whereby mutant CSF-1R recombinant cells are able to form spheroidsindependent of the CSF-1 ligand. Furthermore the antibodies according tothe invention inhibit (both) human and cynomolgous macrophagedifferentiation, as they inhibit survival of human and cynomolgousmonocytes.

II. Definitions

As used herein, the following terms have the meanings ascribed to thembelow unless otherwise specified.

The human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor; Macrophagecolony-stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ IDNO: 22)) is known since 1986 (Coussens, L., et al., Nature 320 (1986)277-280). CSF-1R is a growth factor and encoded by the c-fmsproto-oncogene (reviewed e.g. in Roth, P. and Stanley, E. R., Curr. Top.Microbiol. Immunol. 181 (1992) 141-67).

CSF-1R is the receptor for CSF-1 (macrophage colony stimulating factor,also called M-CSF) and IL-34 and mediates the biological effects ofthese cytokines (Sherr, C. J., et al., Cell 41 (1985) 665-676 (Lin, H.,et al., Science 320 (2008) 807-811). The cloning of the colonystimulating factor-1 receptor (also called c-fms) was described for thefirst time in Roussel, M. F., et al., Nature 325 (1987) 549-552. In thatpublication, it was shown that CSF-1R had transforming potentialdependent on changes in the C-terminal tail of the protein including theloss of the inhibitory tyrosine 969 phosphorylation which binds Cb1 andthereby regulates receptor down regulation (Lee, P. S., et al., Embo J.18 (1999) 3616-3628).

CSF-1R is a single chain, transmembrane receptor tyrosine kinase (RTK)and a member of the family of immunoglobulin (Ig) motif containing RTKscharacterized by 5 repeated Ig-like subdomains D1-D5 in theextracellular domain (ECD) of the receptor (Wang, Z., et al Molecularand Cellular Biology 13 (1993) 5348-5359). The human CSF-1RExtracellular Domain (CSF-1R-ECD) (SEQ ID NO: 64) comprises all fiveextracellular Ig-like subdomains D1-D5. The human CSF-1R fragment delD4(SEQ ID NO: 65) comprises the extracellular subdomains D1-D3 and D5, butis missing the D4 subdomain. The human CSF-1R fragment D1-D3 (SEQ ID NO:66) comprises the respective subdomains D1-D3. The sequences are listedwithout the signal peptide MGSGPGVLLL LLVATAWHGQ G (SEQ ID NO: 67).

The intracellular protein tyrosine kinase domain is interrupted by aunique insert domain that is also present in the other related RTK classIII family members that include the platelet derived growth factorreceptors (PDGFR), stem cell growth factor receptor (c-Kit) andfins-like cytokine receptor (FLT3). In spite of the structural homologyamong this family of growth factor receptors, they have distincttissue-specific functions.

CSF-1R is mainly expressed on cells of the monocytic lineage and in thefemale reproductive tract and placenta. In addition expression of CSF-1Rhas been reported in Langerhans cells in skin, a subset of smooth musclecells (Inaba, T., et al., J. Biol. Chem. 267 (1992) 5693-5699), B cells(Baker, A. H., et al., Oncogene 8 (1993) 371-378) and microglia (Sawada,M., et al., Brain Res. 509 (1990) 119-124). Cells with mutant humanCSF-1R ((SEQ ID NO: 23) are known to proliferate independently of ligandstimulation.

As used herein, “binding to human CSF-1R” or “specifically binding tohuman CSF-1R” refers to an antibody specifically binding to the humanCSF-1R antigen with a binding affinity of KD-value of 1.0×10⁻⁸ mol/l orlower at 35° C., in one embodiment of a KD-value of 1.0×10⁻⁹ mail orlower at 35° C. The binding affinity is determined with a standardbinding assay at 35° C., such as surface plasmon resonance technique(BIAcore®, GE-Healthcare Uppsala, Sweden) A method for determining theKD-value of the binding affinity is described in Example 9. Thus an“antibody binding to human CSF-1R” as used herein refers to an antibodyspecifically binding to the human CSF-1R antigen with a binding affinityof KD 1.0×10⁻⁸ mol/l or lower (preferably 1.0×10⁻⁸ mol/l-1.0×10⁻¹²mol/l) at 35° C., preferably of a KD 1.0×10⁻⁹ mol/l or lower at 35° C.(preferably 1.0×10⁻⁹ mol/l-1.0×10⁻¹² mol/l).

The “binding to human CSF-1R fragment delD4 (SEQ ID NO: 65) and to humanCSF-1R Extracellular Domain (SEQ ID NO: 64)” as used herein is measuredby a Surface Plasmon Resonance assay (Biacore assay) as described inExample 4. The human CSF-1R fragment delD4 (SEQ ID NO: 65) or humanCSF-1R Extracellular Domain (SEQ ID NO: 64), respectively, are capturedto the surface (each to a separate surface) and the test antibodies wereadded (each in a separate measurement), and the respective bindingsignals (Response Units (RU)) were determined. Reference signals (blanksurface) were subtracted. If signals of nonbinding test antibodies wereslightly below 0 the values were set as 0. Then the ratio of therespective binding signals (binding signal (RU) to human CSF-1R fragmentdelD4/binding signal (RU) to human CSF-1R Extracellular Domain(CSF-1R-ECD)) is determined. The antibodies according to the inventionhave a ratio of the binding signals (RU(delD4)/RU(CSF-1R-ECD) of 1:50 orlower, preferably of 1:100 or lower (the lower included end is 0 (e.g.if the RU is 0, then the ratio is 0:50 or 0:100)).

This means that such anti-CSF-1R antibodies according to the inventiondo not bind to the human CSF-1R fragment delD4 (like the anti-CCR5antibody m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18 Aug. 2004 atDSMZ) and have binding signals for binding to the human CSF-1R fragmentdelD4 in the range of the anti-CCR5 antibody m<CCR5>Pz03.1C5, which arebelow 20 RU (Response Units), preferably below 10 RU in a SurfacePlasmon Resonance (BIAcore) assay as shown in Example 4.

The term “binding to human CSF-1R fragment D1-D3” refers to a bindingaffinity determination by a Surface Plasmon Resonance assay (Biacoreassay). The test antibody is captured to the surface and the humanCSF-1R fragment D1-D3 (SEQ ID NO: 66) was added and the respectivebinding affinities were determined. The term “not binding to humanCSF-1R fragment D1-D3” denotes that in such an assay the detected signalwas in the area of no more than 1.2 fold of background signal andtherefore no significant binding could be detected and no bindingaffinity could he determined (see Example 10).

The term “antibody” encompasses the various forms of antibodiesincluding but not being limited to whole antibodies, antibody fragments,human antibodies, humanized antibodies, chimeric antibodies, T cellepitope depleted antibodies, and further genetically engineeredantibodies as long as the characteristic properties according to theinvention are retained. “Antibody fragments” comprise a portion of afull length antibody, preferably the variable domain thereof, or atleast the antigen binding site thereof. Examples of antibody fragmentsinclude diabodies, single-chain antibody molecules, and multispecificantibodies formed from antibody fragments. scFv antibodies are, e.g.,described in Houston, J. S., Methods in Enzymol, 203 (1991) 46-88). Inaddition, antibody fragments comprise single chain polypeptides havingthe characteristics of a V_(H) domain binding to CSF-1R, namely beingable to assemble together with a V_(L) domain, or of a V_(L) domainbinding to CSF-1R, namely being able to assemble together with a V_(H)domain to a functional antigen binding site and thereby providing theproperty.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from mouse and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a mouse variable region and a human constant region areespecially preferred. Such rat/human chimeric antibodies are the productof expressed immunoglobulin genes comprising DNA segments encoding ratimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of “chimeric antibodies”encompassed by the present invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies.” Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., Morrison, S. L., et al., Proc.Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 andU.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See e.g.Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Optionally the framework region canbe modified by further mutations. Also the CDRs can be modified by oneor more mutations to generate antibodies according to the invention e.g.by mutagenesis based upon molecular modeling as described by Riechmann,L., et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl.Acad. Sci. USA 86 (1989) 10029-10033, or others. Particularly preferredCDRs correspond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. A “humanized version of an antibodyaccording to the invention” (which is e.g. of mouse origin) refers to anantibody, which is based on the mouse antibody sequences in which theV_(H) and V_(L) are humanized by standard techniques (including CDRgrafting and optionally subsequent mutagenesis of certain amino acids inthe framework region and the CDRs). Preferably such humanized version ischimerized with a human constant region (see e.g. Sequences SEQ IDNO:57-61).

Other forms of “humanized antibodies” encompassed by the presentinvention are those in which the constant region has been additionallymodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding.

In the following examples the terms “Mab” or “muMab” refer to murinemonoclonal antibodies such as Mab 2F11 or Mab 2E10, whereas the term“hMab” refers to humanized monoclonal versions of such murine antibodiessuch as hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7 hMab 2F11-f12, etc.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits., et al., Nature 362 (1993) 255-258; Brueggemann,M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also beproduced in phage display libraries (Hoogenboom, H. R., and Winter, G.J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol.222 (1991) 581-597). The techniques of Cole, et al and Boemer, et al.,are also available for the preparation of human monoclonal antibodies(Cole, S. P. C., et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991)86-95). As already mentioned for chimeric and humanized antibodiesaccording to the invention the term “human antibody” as used herein alsocomprises such antibodies which are modified in the constant region togenerate the properties according to the invention, especially in regardto C1q binding and/or FcR binding, e.g. by “class switching” i.e. changeor mutation of Fc parts (e.g. from IgG1 to IgG4 and/or IgG1/IgG4mutation).

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications”, nucleotide andamino acid sequence modifications which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-CSF-1Rantibody can he preferably replaced with another amino acid residue fromthe same side chain family.

Amino acid substitutions can be performed by mutagenesis based uponmolecular modeling as described by Riechmann, L., et al, Nature 332(1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033.

The term “epitope” denotes a protein determinant of human CSF-1R capableof specifically binding to an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually epitopes have specific three dimensionalstructural characteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Preferably an antibody according to the inventionbinds specifically to native and to denatured CSF-1R.

The “variable domain” (variable domain of a light chain (V_(L)),variable domain of a heavy chain (V_(H))) as used herein denotes each ofthe pair of light and heavy chain domains which are involved directly inbinding the antibody to the antigen. The variable light and heavy chaindomains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementary determining regions,CDRs). The framework regions adopt a β-sheet conformation and the CDRsmay form loops connecting the β-sheet structure. The CDRs in each chainare held in their three-dimensional structure by the framework regionsand form together with the CDRs from the other chain the antigen bindingsite. The antibody's heavy and light chain CDR3 regions play aparticularly important role in the binding specificity/affinity of theantibodies according to the invention and therefore provide a furtherobject of the invention.

The term “antigen-binding portion of an antibody” when used herein referto the amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. “Framework” or “FR” regions are those variable domain regionsother than the hypervariable region residues as herein defined.Therefore, the light and heavy chain variable domains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding and defines the antibody'sproperties. CDR and FR regions are determined according to the standarddefinition of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991) and/or those residues from a “hypervariable loop”.

The “Fc part” or “Fc portion” of an antibody is not involved directly inbinding of an antibody to an antigen, hut exhibit various effectorfunctions. A “Fc part of an antibody” is a term well known to theskilled artisan and defined on the basis of papain cleavage ofantibodies. Depending on the amino acid sequence of the constant regionof their heavy chains, antibodies or immunoglobulins are divided in theclasses: IgA, IgD, IgE, IgG and IgM, and several of these may be furtherdivided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4,IgA1, and IgA2. According to the heavy chain constant regions thedifferent classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. The Fc part of an antibody is directly involved in ADCC(antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity) based on complement activation, C1qbinding and Fc receptor binding. Complement activation (CDC) isinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. While the influence of an antibody on thecomplement system is dependent on certain conditions, binding to C1q iscaused by defined binding sites in the Fc part. Such binding sites areknown in the state of the art and described e.g. by Boackle, R. J., etal., Nature 282 (1979) 742-743, Lukas, T. J., et al., J. Immunol. 127(1981) 2555-2560, Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16(1979) 907-917, Burton, D. R., et al., Nature 288 (1980) 338-344,Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie, E.E., et al., J. Immunol.164 (2000) 4178-4184, Hezareh, M., et al., J.Virology 75 (2001) 12161-12168, Morgan, A., et al., Immunology 86 (1995)319-324, EP 0307434. Such binding sites are e.g. L234, L235, D270, N297,E318, K320, K322, P331 and P329 (numbering according to EU index ofKabat, E. A., see below). Antibodies of subclass IgG1, IgG2 and IgG3usually show complement activation and C1q and C3 binding, whereas IgG4do not activate the complement system and do not bind C1q and C3.

In one embodiment the antibody according to the invention comprises a Fcpart derived from human origin and preferably all other parts of thehuman constant regions. As used herein the term “Fc part derived fromhuman origin” denotes a Fc part which is either a Fc part of a humanantibody of the subclass IgG1, IgG2, IgG3 or IgG4, preferably a Fc partfrom human IgG1 subclass, a mutated Fc part from human IgG1 subclass(preferably with a mutation on L234A+L235A), a Fc part from human IgG4subclass or a mutated Fc part from human IgG4 subclass (preferably witha mutation on S228P). Mostly preferred are the human heavy chainconstant regions of SEQ ID NO: 58 (human IgG1 subclass), SEQ ID NO: 59(human IgG1 subclass with mutations L234A and L235A), SEQ ID NO: 60human IgG4 subclass), or SEQ ID NO: 61 (human IgG4 subclass withmutation S228P).

The terms “nucleic acid” or “nucleic acid molecule”, as used herein, areintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount encompasses an amount in which anytoxic or detrimental effects of the substance/molecule are outweighed bythe therapeutically beneficial effects. A therapeutically effectiveamount also encompasses an amount sufficient to confer benefit, e.g.,clinical benefit.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount would be less than thetherapeutically effective amount. A prophylactically effective amountencompasses an amount sufficient to confer benefit, e.g., clinicalbenefit.

In the case of pre-cancerous, benign, early or late-stage tumors, thetherapeutically effective amount of the angiogenic inhibitor may reducethe number of cancer cells; reduce the primary tumor size; inhibit(i.e., slow to some extent and preferably stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent andpreferably stop) tumor metastasis; inhibit or delay, to some extent,tumor growth or tumor progression; and/or relieve to some extent one ormore of the symptoms associated with the disorder. To the extent thedrug may prevent growth and/or kill existing cancer cells, it may becytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can,for example, be measured by assessing the duration of survival, time todisease progression (TTP), the response rates (RR), duration ofresponse, and/or quality of life.

To “reduce” or “inhibit” is to decrease or reduce an activity, function,and/or amount as compared to a reference. In certain embodiments, by“reduce” or “inhibit” is meant the ability to cause an overall decreaseof 20% or greater. In another embodiment, by “reduce” or “inhibit” ismeant the ability to cause an overall decrease of 50% or greater. In yetanother embodiment, by “reduce” or “inhibit” is meant the ability tocause an overall decrease of 75%, 85%, 90%, 95%, or greater. Reduce orinhibit can refer to the symptoms of the disorder being treated, thepresence or size of metastases, the size of the primary tumor, or thesize or number of the blood vessels in angiogenic disorders.

A “disorder” is any condition that would benefit from treatmentincluding, but not limited to, chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Disorders include cancer (including metastaticcancer), bone disorders (including, e.g., bone loss such asosteoporosis), and inflammatory disorders.

III. Antibodies and Methods of the Invention

The invention comprises an antibody binding to human CSF-1R, wherein theantibody binds to human CSF-1R fragment delD4 (comprising theextracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) and to humanCSF-1R Extracellular Domain (CSF-1R-ECD) (comprising the extracellularsubdomains D1-D5) (SEQ ID NO: 64) with a ratio of 1:50 or lower.

The invention further comprises an antibody comprising a heavy chainvariable domain CDR3 region comprising SEQ ID NO: 1, SEQ ID NO: 9, SEQID NO:23, SEQ ID NO:31, SEQ NO:39, SEQ ID NO:47 or SEQ ID NO:55.

The invention further comprises an antibody wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ ID NO:15 and        the light chain variable domain comprises SEQ ID NO:16; or a        humanized version thereof.

The invention further comprises an antibody wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ ID NO:15 and        the light chain variable domain comprises SEQ ID NO:16;    -   c) the heavy chain variable domain comprises SEQ ID NO:75 and        the light chain variable domain comprises SEQ ID NO:76;    -   d) the heavy chain variable domain comprises SEQ ID NO:83 and        the light chain variable domain comprises SEQ ID NO:84;

or a humanized version thereof.

The invention further comprises an antibody wherein

-   -   the heavy chain variable domain comprises SEQ NO:7 and the light        chain variable domain comprises SEQ NO:8, or a humanized version        thereof.

In one embodiment the antibody comprises

-   -   a) a heavy chain variable domain comprising SEQ ID NO:23 and a        light chain variable domain comprising SEQ ID NO:24, or    -   b) a heavy chain variable domain comprising SEQ ID NO:31 and a        light chain variable domain comprising SEQ ID NO:32, or    -   c) a heavy chain variable domain comprising SEQ ID NO:39 and a        light chain variable domain comprising SEQ ID NO:40, or    -   d) a heavy chain variable domain comprising SEQ ID NO:47 and a        light chain variable domain comprising SEQ ID NO:48, or    -   e) a heavy chain variable domain comprising SEQ ID NO:55 and a        light chain variable domain comprising SEQ ID NO:56.

In one embodiment the antibody comprises

-   -   a) a heavy chain variable domain comprising SEQ ID NO:23 and a        light chain variable domain comprising SEQ ID NO:24, or    -   b) a heavy chain variable domain comprising SEQ ID NO:31 and a        light chain variable domain comprising SEQ ID NO:32, or    -   c) a heavy chain variable domain comprising SEQ NO:39 and a        light chain variable domain comprising SEQ ID NO:40, or    -   d) a heavy chain variable domain comprising SEQ ID NO:47 and a        light chain variable domain comprising SEQ ID NO:48.

In one embodiment the antibody comprises

-   -   a heavy chain variable domain comprising SEQ ID NO:23 and a        light chain variable domain comprising SEQ ID NO:24, or

In one embodiment the antibody comprises

-   -   a heavy chain variable domain comprising SEQ ID NO:31 and a        light chain variable domain comprising SEQ ID NO:32.

In one embodiment the antibody comprises

-   -   a heavy chain variable domain comprising SEQ ID NO:39 and a        light chain variable domain comprising SEQ ID NO:40.

In one embodiment the antibody comprises

-   -   a heavy chain variable domain comprising SEQ ID NO:47 and a        light chain variable domain comprising SEQ ID NO:48.

The invention further comprises an antibody wherein

-   -   the heavy chain variable domain comprises SEQ ID NO: 15 and the        light chain variable domain comprises SEQ ID NO:16, or a        humanized version thereof.

The invention further comprises an antibody wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:75 and the        light chain variable domain comprises SEQ ID NO:76;

or a humanized version thereof.

The invention further comprises an antibody wherein

-   -   the heavy chain variable domain comprises SEQ NO:83 and the        light chain variable domain comprises SEQ ID NO:84;

or a humanized version thereof.

The invention further comprises an antibody, wherein

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:1, a CDR2 region comprising SEQ ID NO: 2,        and a CDR1 region comprising SEQ ID NO:3, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO: 4,        a CDR2 region comprising SEQ ID NO:5, and a CDR1 region        comprising SEQ ID NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 9, a CDR2 region comprising SEQ ID NO: 10,        and a CDR1 region comprising SEQ ID NO: 11, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        12, a CDR2 region comprising SEQ ID NO: 13, and a CDR1 region        comprising SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO: 19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ ID NO: 51, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:52, a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54.

The invention further comprises an antibody, wherein

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:1, a CDR2 region comprising SEQ ID NO: 2,        and a CDR1 region comprising SEQ ID NO:3, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO: 4,        a CDR2 region comprising SEQ ID NO:5, and a CDR1 region        comprising SEQ ID NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 9, a CDR2 region comprising SEQ ID NO: 10,        and a CDR1 region comprising SEQ ID NO: 11, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:12,        a CDR2 region comprising SEQ NO: 13, and a CDR1 region        comprising SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region        comprising SEQ NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ NO:46,    -   g) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ ID NO: 51, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:52, a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54;    -   h) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:69, a CDR2 region comprising SEQ ID NO: 70,        and a CDR1 region comprising SEQ ID NO:71, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        72, a CDR2 region comprising SEQ ID NO:73, and a CDR1 region        comprising SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 77, a CDR2 region comprising SEQ ID NO:        78, and a CDR1 region comprising SEQ ID NO: 79, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:80, a CDR2 region comprising SEQ ID NO: 81, and a CDR1 region        comprising SEQ ID NO: 82.

In one embodiment the antibody comprises

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:69, a CDR2 region comprising SEQ ID NO: 70,        and a CDR1 region comprising SEQ ID NO:71, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        72, a CDR2 region comprising SEQ NO:73, and a CDR1 region        comprising SEQ ID NO:74, or    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 77, a CDR2 region comprising SEQ ID NO:        78, and a CDR1 region comprising SEQ ID NO: 79, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:80, a CDR2 region comprising SEQ ID NO: 81, and a CDR1 region        comprising SEQ ID NO: 82.

In one embodiment the antibody comprises

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO: 19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46, or    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ ID NO: 51, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:52, a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54.

In one embodiment the antibody comprises

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46.

In one embodiment the antibody comprises

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22,

In one embodiment the antibody comprises

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30.

In one embodiment the antibody comprises

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37 and a CDR1 region        comprising SEQ ID NO: 38.

In one embodiment the antibody comprises

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46.

In one embodiment the antibody binding to human CSF-1R binds to humanCSF-1R fragment delD4 (SEQ ID NO: 65) and to human CSF-1R-ECD (SEQ IDNO: 64) with a ratio of 1:50 or lower, further does not bind to humanCSF-1R fragment D1-D3 (SEQ ID NO: 66).

One embodiment of the invention is a screening method for selectingantibodies according to the invention comprising the following steps:

-   -   a) determining the binding signal (Response Units (RU)) of        anti-CSF-1R antibodies to human CSF-1R fragment delD4 (SEQ ID        NO: 65) and to human CSF1R Extracellular Domain (CSF-1R-ECD)        (SEQ ID NO: 64) by a Surface Plasmon Resonance assay (Biacore        assay).    -   h) selecting antibodies with ratio of the binding signals (human        CSF-1R fragment delD4/human CSF-1R Extracellular Domain        (CSF-1R-ECD)) of 50:1 or lower.

In one embodiment the determination is performed at 25° C.

In one embodiment the screening method comprises as further steps themeasuring of the binding of anti-CSF-1R antibodies to human CSF-1Rfragment D1-D3 (SEQ ID NO: 66) (D1-D3) and the selecting of antibodieswhich show no binding to said fragment.

In one embodiment the antibodies according to the invention inhibitCSF-1 binding to CSF-1R. In one embodiment with an IC50 of 200 ng/ml orlower, in one embodiment with an IC50 of 50 ng/ml or lower. The IC50 ofinhibition of CSF-1 binding to CSF-1R can be determined as shown inExample 2.

In one embodiment the antibodies according to the invention inhibitCSF-1-induced CSF-1R phosphorylation (in NIH3T3-CSF-1R recombinantcells).

In one embodiment with an IC50 of 800 ng/ml or lower, in one embodimentwith an IC50 of 600 ng/ml or lower, in one embodiment with an IC50 of250 ng/ml or lower. The IC50 of CSF-1-induced CSF-1R phosphorylation canbe determined as shown in Example 3.

In one embodiment the antibodies according to the invention inhibit thegrowth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID No:62). In one embodiment with an IC50 of 10 μg/ml or lower, in oneembodiment with an IC50 of 5 μg/ml or lower, in one embodiment with anIC50 of 2 μg/ml or lower. In one embodiment with an IC30 of 10 μg/ml orlower, in one embodiment with an IC30 of 5 μg/ml or lower, in oneembodiment with an IC30 of 2 μg/ml or lower. The IC50 value, the IC30value or the % growth inhibition is determined as shown in Example 5.

In one embodiment the antibodies according to the invention inhibit thegrowth of recombinant NIH3T3 cells expressing human mutant CSF-1R L301SY969F (SEQ ID No: 63). In one embodiment with an IC50 of 15 μg/ml orlower, in one embodiment with an IC50 of 10 μg/ml or lower. In oneembodiment with an IC30 of 10 μg/ml or lower, in one embodiment with anIC50 of 5 μg/ml ng/ml or lower; in one embodiment with an IC50 of 2μg/ml or lower. The IC50 value, the IC30 value or the % growthinhibition is determined as shown in Example 5.

In one embodiment the antibodies according to the invention inhibit thegrowth of BeWo tumor cells (ATCC CCL-98) by 65% or more (at an antibodyconcentration of 10 μg/ml; and as compared to the absence of antibody).The % growth inhibition is determined as shown in Example 8. E.g. Mab2F11 shows a growth inhibition of BeWo tumor cells of 70%.

In one embodiment the antibodies according to the invention inhibit(both) human and cynomolgous macrophage differentiation (which isindicated by the inhibition of the survival of human and cynomolgousmonocytes as shown in Examples 7 and 8). In one embodiment theantibodies according to the invention inhibit the survival of humanmonocytes with an IC50 of 0.15 μg/ml or lower, in on embodiment with anIC50 of 0.10 μg/ml or lower. The inhibition of the survival, of humanmonocytes is determined as shown in Example 7. In one embodiment theantibodies according to the invention inhibit the survival ofcynomolgous monocytes by 80% or more, in one embodiment by 90% or more(at an antibody concentration of 5 μg/ml; and as compared to the absenceof antibody). The inhibition of the survival of human monocytes isdetermined as shown in Example 8.

A further embodiment of the invention is a method for the production ofan antibody against CSF-1R wherein the sequence of a nucleic acidencoding the heavy chain of a human IgG1 class antibody binding to humanCSF-1R according to the invention said modified nucleic acid and thenucleic acid encoding the light chain of said antibody are inserted intoan expression vector, said vector is inserted in a eukaryotic host cell,the encoded protein is expressed and recovered from the host cell or thesupernatant.

The antibodies according to the invention are preferably produced byrecombinant means. Therefore the antibody is preferably an isolatedmonoclonal antibody. Such recombinant methods are widely known in thestate of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody polypeptideand usually purification to a pharmaceutically acceptable purity. Forthe protein expression, nucleic acids encoding light and heavy chains orfragments thereof are inserted into expression vectors by standardmethods. Expression is performed in appropriate prokaryotic oreukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293cells, COS cells, yeast, or E.coli cells, and the antibody is recoveredfrom the cells (supernatant or cells after lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl handing, column chromatography,agarose gel electrophoresis, and others well known in the art. SeeAusubel, F., et al., ed. Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York (1987).

The monoclonal antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies are readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

In some embodiments, the antibody according to the invention is of humanIgG1 subclass or of human IgG4 subclass. In one embodiment the antibodyaccording to the invention is of human IgG1 subclass. In one embodimentthe antibody according to the invention is of human IgG4 subclass.

In one embodiment the constant chains are of human origin. Such constantchains are well known in the state of the art and e.g. described byKabat, E. A., (see e.g., Johnson, G. and Wu, T. T., Nucleic Acids Res.28 (2000) 214-218). For example, a useful human heavy chain constantregion comprises an amino acid sequence of SEQ ID NO: 58. For example, auseful human light chain constant region comprises an amino acidsequence of a kappa-light chain constant region of SEQ ID NO: 57.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ ID NO:15 and        the light chain variable domain comprises SEQ ID NO:16;    -   or a humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8,    -   b) the heavy chain variable domain comprises SEQ ID NO:15 and        the light chain variable domain comprises SEQ ID NO:16;    -   c) the heavy chain variable domain comprises SEQ ID NO:75 and        the light chain variable domain comprises SEQ ID NO:76;    -   d) the heavy chain variable domain comprises SEQ ID NO:83 and        the light chain variable domain comprises SEQ ID NO:84;

or a humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:7 and the        light chain variable domain comprises SEQ ID NO:8, or a        humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:23 and        the light chain variable domain comprises SEQ ID NO:24, or    -   b) the heavy chain variable domain comprises SEQ NO:31 and the        light chain variable domain comprises SEQ ID NO:32, or    -   c) the heavy chain variable domain comprises SEQ ID NO:39 and        the light chain variable domain comprises SEQ ID NO:40, or    -   d) the heavy chain variable domain comprises SEQ ID NO:47 and        the light chain variable domain comprises SEQ ID NO:48, or    -   e) the heavy chain variable domain comprises SEQ ID NO:55 and        the light chain variable domain comprises SEQ ID NO:56.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises SEQ ID NO:23 and        the light chain variable domain comprises SEQ ID NO:24, or    -   b) the heavy chain variable domain comprises SEQ ID NO:31 and        the light chain variable domain comprises SEQ ID NO:32, or    -   c) the heavy chain variable domain comprises SEQ ID NO:39 and        the light chain variable domain comprises SEQ NO:40, or    -   d) the heavy chain variable domain comprises SEQ ID NO:47 and        the light chain variable domain comprises SEQ ID NO:48.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:23 and the        light chain variable domain comprises SEQ ID NO:24, or

Another aspect of the invention is an antibody binding to human CSF-1R,wherein the heavy chain variable domain comprises SEQ ID NO:31 and thelight chain variable domain comprises SEQ ID NO:32.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:39 and the        light chain variable domain comprises SEQ ID NO:40,

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:47 and the        light chain variable domain comprises SEQ ID NO:48.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:15 and the        light chain variable domain comprises SEQ ID NO:16, or a        humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:75 and the        light chain variable domain comprises SEQ ID NO:76;

or a humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises SEQ ID NO:83 and the        light chain variable domain comprises SEQ ID NO:84;

or a humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:1, a CDR2 region comprising SEQ ID NO: 2,        and a CDR1 region comprising SEQ ID NO:3, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO: 4,        a CDR2 region comprising SEQ ID NO:5, and a CDR1 region        comprising SEQ ID NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 9, a CDR2 region comprising SEQ ID NO: 10,        and a CDR1 region comprising SEQ ID NO: 11, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:12,        a CDR2 region comprising SEQ ID NO: 13, and a CDR1 region        comprising SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46,    -   g) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ ID NO: 51, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:52, a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54;    -   h) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:69, a CDR2 region comprising SEQ ID NO: 70,        and a CDR1 region comprising SEQ ID NO:71, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        72, a CDR2 region comprising SEQ ID NO:73, and a CDR1 region        comprising SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 77, a CDR2 region comprising SEQ ID NO:        78, and a CDR1 region comprising SEQ ID NO: 79, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:80, a CDR2 region comprising SEQ ID NO: 81, and a CDR1 region        comprising SEQ ID NO: 82.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO: 19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46, or    -   e) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 49, a CDR2 region comprising SEQ ID NO:        50, and a CDR1 region comprising SEQ ID NO: 51, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:52, a CDR2 region comprising SEQ ID NO: 53, and a CDR1 region        comprising SEQ ID NO: 54.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   a) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO: 19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ NO:21, and a CDR1 region        comprising SEQ ID NO:22, or    -   b) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30, or    -   c) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38, or    -   d) the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 17, a CDR2 region comprising SEQ ID NO:        18, and a CDR1 region comprising SEQ ID NO:19, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO: 20, a CDR2 region comprising SEQ ID NO:21, and a CDR1 region        comprising SEQ ID NO:22.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 25, a CDR2 region comprising SEQ ID NO:        26, and a CDR1 region comprising SEQ ID NO: 27, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:28, a CDR2 region comprising SEQ ID NO: 29, and a CDR1 region        comprising SEQ ID NO: 30.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO: 33, a CDR2 region comprising SEQ ID NO:        34, and a CDR1 region comprising SEQ ID NO: 35, and the light        chain variable domain comprises a CDR3 region comprising SEQ ID        NO:36, a CDR2 region comprising SEQ ID NO: 37, and a CDR1 region        comprising SEQ ID NO: 38.

Another aspect of the invention is an antibody binding to human CSF-1R,wherein

-   -   the heavy chain variable domain comprises a CDR3 region        comprising SEQ ID NO:41, a CDR2 region comprising SEQ ID NO: 42,        and a CDR1 region comprising SEQ ID NO:43, and the light chain        variable domain comprises a CDR3 region comprising SEQ ID NO:        44, a CDR2 region comprising SEQ ID NO:45, and a CDR1 region        comprising SEQ ID NO:46.

The invention comprises a method for the treatment of a patient in needof therapy, comprising administering to the patient a therapeuticallyeffective amount of an antibody according to the invention.

The invention comprises the use of an antibody according to theinvention for therapy.

One preferred embodiment of the invention are the CSF-1R antibodies ofthe present invention for use in the treatment of “CSF-1R mediateddiseases” or the CSF-1R antibodies of the present invention for use forthe manufacture of a medicament in the treatment of “CSF-1R mediateddiseases”, which can be described as follows:

There are 3 distinct mechanisms by which CSF-1R signaling is likelyinvolved in tumor growth and metastasis. The first is that expression ofCSF-ligand and receptor has been found in tumor cells originating in thefemale reproductive system (breast, ovarian, endometrium, cervical)(Scholl, S. M., et al, J. Natl. Cancer Inst. 86 (1994) 120-126;Kacinski, B. M., Mol. Reprod. Dev. 46 (1997) 71-74; Ngan, H. Y., et al.,Eur. J. Cancer 35 (1999) 1546-1550; Kirma, N., et al., Cancer Res 67(2007) 1918-1926) and the expression has been associated with breastcancer xenograft growth as well as poor prognosis in breast cancerpatients. Two point mutations were seen in CSF-1R in about 10-20% ofacute myelocytic leukemia, chronic myelocytic leukemia andmyelodysplasia patients tested in one study, and one of the mutationswas found to disrupt receptor turnover (Ridge, S. A., et al., Proc.Natl. Acad. Sci USA 87 (1990) 1377-1380). However the incidence of themutations could not be confirmed in later studies (Abu-Duhier, F. M., etal., Br. J. Haematol. 120 (2003) 464-470). Mutations were also found insome cases of hepatocellular cancer (Yang, D. H., et al., HepatobiliaryPancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis(Abu-Duhier, F. M., et al., Br. J. Haematol. 120 (2003) 464-470).Recently, in the GDM-1 cell line derived from a patient withmyelomonoblastic leukemia the Y571D mutation in CSF-1R was identified(Chase, A., et al., Leukemia 23 (2009) 358-364).

Pigmented villonodular synovitis (PVNS) and Tenosynovial Giant celltumors (TGCT) can occur as a result of a translocation that fuses theM-CSF gene to a collagen gene COL6A3 and results in overexpression ofM-CSF (West, R. B., et al., Proc. Natl. Acad. Sci. USA 103 (2006)690-695). A landscape effect is proposed to be responsible for theresulting tumor mass that consists of monocytic cells attracted by cellsthat express M-CSF. TGCTs are smaller tumors that can be relativelyeasily removed from fingers where they mostly occur. PVNS is moreaggressive as it can recur in large joints and is not as easilycontrolled surgically.

The second mechanism is based on blocking signaling through M-CSF/CSF-1Rat metastatic sites in bone which induces osteoclastogenesis, boneresorption and osteolytic bone lesions. Breast, multiple myeloma andlung cancers are examples of cancers that have been found to metastasizeto the bone and cause osteolytic bone disease resulting in skeletalcomplications. M-CSF released by tumor cells and stroma induces thedifferentiation of hematopoietic myeloid monocyte progenitors to matureosteoclasts in collaboration with the receptor activator of nuclearfactor kappa-B ligand-RANKL. During this process, M-CSF acts as apermissive factor by giving the survival signal to osteoclasts (Tanaka,S., et al., J. Clin. Invest. 91 (1993) 257-263). Inhibition of CSF-1Ractivity during osteoclast differentiation and maturation with aanti-CSF-1R antibody is likely to prevent unbalanced activity ofosteoclasts that cause osteolytic disease and the associated skeletalrelated events in metastatic disease. Whereas breast, lung cancer andmultiple myeloma typically result in osteolytic lesions, metastasis tothe bone in prostate cancer initially has an osteoblastic appearance inwhich increased bone forming activity results in ‘woven bone’ which isdifferent from typical lamellar structure of normal bone. During diseaseprogression bone lesions display a significant osteolytic component aswell as high serum levels of bone resorption and suggests thatanti-resorptive therapy may be useful. Bisphosphonates have been shownto inhibit the formation of osteolytic lesions and reduced the number ofskeletal-related events only in men with hormone-refractory metastaticprostate cancer but at this point their effect on osteoblastic lesionsis controversial and bisphosphonates have not been beneficial inpreventing bone metastasis or hormone responsive prostate cancer todate. The effect of anti-resorptive agents in mixedosteolytic/osteoblastic prostate cancer is still being studied in theclinic (Choueiri, M. B., et al., Cancer Metastasis Rev. 25 (2006)601-609; Vessella R. L. and Corey, E., Clin. Cancer Res. 12 (20 Pt 2)(2006) 6285s-6290s).

The third mechanism is based on the recent observation that tumorassociated macrophages (TAM) found in solid tumors of the breast,prostate, ovarian and cervical cancers correlated with poor prognosis(Bingle, L., et al., J. Pathol. 196 (2002) 254-265; Pollard, J. W., Nat.Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to the tumor byM-CSF and other chemokines. The macrophages can then contribute to tumorprogression through the secretion of angiogenic factors, proteases andother growth factors and cytokines and may be blocked by inhibition ofCSF-1R signaling. Recently it was shown by Zins et al (Zins, K., et al.,Cancer Res. 67 (2007) 1038-1045) that expression of siRNA of Tumornecrosis factor alpha (TNF alpha), M-CSF or the combination of bothwould reduce tumor growth in a mouse xenograft model between 34% and 50%after intratumoral injection of the respective siRNA. SiRNA targetingthe TNF alpha secreted by the human SW620 cells reduced mouse M-CSFlevels and led to reduction of macrophages in the tumor. In additiontreatment of MCF7 tumor xenografts with an antigen binding fragmentdirected against M-CSF did result in 40% tumor growth inhibition,reversed the resistance to chemotherapeutics and improved survival ofthe mice when given in combination with chemotherapeutics (Paulus, P.,et al., Cancer Res. 66 (2006) 4349-4356).

TAMs are only one example of an emerging link between chronicinflammation and cancer. There is additional evidence for a link betweeninflammation and cancer as many chronic diseases are associated with anincreased risk of cancer, cancers arise at sites of chronicinflammation, chemical mediators of inflammation are found in manycancers; deletion of the cellular or chemical mediators of inflammationinhibits development of experimental cancers and long-term use ofanti-inflammatory agents reduce the risk of some cancers. A link tocancer exists for a number of inflammatory conditions among—thoseH.pylori induced gastritis for gastric cancer, Schistosomiasis forbladder cancer, HHVX for Kaposi's sarcoma, endometriosis for ovariancancer and prostatitis for prostate cancer (Balkwill, F., et al., CancerCell 7 (2005) 211-217). Macrophages are key cells in chronicinflammation and respond differentially to their microenvironment. Thereare two types of macrophages that are considered extremes in a continuumof functional states: M1 macrophages are involved in Type 1 reactions.These reactions involve the activation by microbial products andconsequent killing of pathogenic microorganisms that result in reactiveoxygen intermediates. On the other end of the extreme are M2 macrophagesinvolved in Type 2 reactions that promote cell proliferation, tuneinflammation and adaptive immunity and promote tissue remodeling,angiogenesis and repair (Mantovani, A., et al., Trends Immunol. 25(2004) 677-686). Chronic inflammation resulting in established neoplasiais usually associated with M2 macrophages. A pivotal cytokine thatmediates inflammatory reactions is TNF alpha that true to its name canstimulate anti-tumor immunity and hemorrhagic necrosis at high doses buthas also recently been found to be expressed by tumor cells and actingas a tumor promoter (Zins, K., et al., Cancer Res. 67 (2007) 1038-1045;Balkwill, F., Cancer Metastasis Rev. 25 (2006) 409-416). The specificrole of macrophages with respect to the tumor still needs to be betterunderstood including the potential spatial and temporal dependence ontheir function and the relevance to specific tumor types.

Thus one embodiment of the invention are the CSF-1R antibodies of thepresent invention for use in the treatment of cancer. The term “cancer”as used herein may be, for example, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra., cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma, lymphoma, lymphocytic leukemia, including refractoryversions of any of the above cancers, or a combination of one or more ofthe above cancers. Preferably such cancer is a breast cancer, ovariancancer, cervical cancer, lung cancer or prostate cancer. Preferably suchcancers are further characterized by CSF-1 or CSF-1R expression oroverexpression. One further embodiment the invention are the CSF-1Rantibodies of the present invention for use in the simultaneoustreatment of primary tumors and new metastases.

Thus another embodiment of the invention are the CSF-1R antibodies ofthe present invention for use in the treatment of periodontitis,histiocytosis X. osteoporosis, Paget's disease of bone (PDB), bone lossdue to cancer therapy, periprosthetic osteolysis, glucocorticoid-inducedosteoporosis, rheumatoid arthritis, psiratic arthritis, osteoarthritis,inflammatory arthridities, and inflammation.

Rabello, D., et al., Biochem. Biophys. Res. Commun. 347 (2006) 791-796has demonstrated that SNPs in the CSF1 gene exhibited a positiveassociation with aggressive periodontitis: an inflammatory disease ofthe periodontal tissues that causes tooth loss due to resorption of thealveolar bone.

Histiocytosis X (also called Langerhans cell histiocytosis, LCH) is aproliferative disease of Langerhans dendritic cells that appear todifferentiate into osteoclasts in bone and extra osseous LCH lesions.Langerhans cells are derived from circulating monocytes. Increasedlevels of M-CSF that have been measured in sera and lesions where foundto correlate with disease severity (da Costa, C. E., et al., J. Exp.Med. 201 (2005) 687-693). The disease occurs primarily in a pediatricpatient population and has to be treated with chemotherapy when thedisease becomes systemic or is recurrent.

The pathophysiology of osteoporosis is mediated by loss of bone formingosteoblasts and increased osteoclast dependent bone resorption.Supporting data has been described by Cenci et al showing that ananti-M-CSF antibody injection preserves bone density and inhibits boneresorption in ovariectomized mice (Cenci, S., et al., J. Clin. Invest.105 (2000) 1279-1287). Recently a potential link between postmenopausalbone loss due to estrogen deficiency was identified and found that thepresence of TNF alpha producing T-cell affected bone metabolism (Roggia,C., et al., Minerva Med. 95 (2004) 125-132). A possible mechanism couldbe the induction of M-CSF by TNF: alpha in vivo. An important role forM-CSF in TNF-alpha-induced osteoclastogenesis was confirmed by theeffect of an antibody directed against. M-CSF that blocked the TNF alphainduced osteolysis in mice and thereby making inhibitors of CSF-1Rsignaling potential targets for inflammatory arthritis (Kitaura, H., etal., J. Clin. Invest. 115 (2005) 3418-3427).

Paget's disease of bone (PDB) is the second most common bone metabolismdisorder after osteoporosis in which focal abnormalities of increasedbone turnover lead to complications such as bone pain, deformity,pathological fractures and deafness. Mutations in four genes have beenidentified that regulate normal osteoclast function and predisposeindividuals to PDB and related disorders: insertion mutations inTNFRSF11A, which encodes receptor activator of nuclear factor (NT)kappaB (RANK)-a critical regulator of osteoclast function, inactivatingmutations of TNFRSF11B which encodes osteoprotegerin (a decoy receptorfor RANK ligand), mutations of the sequestosome I gene (SQSTM41), whichencodes an important scaffold protein in the NFkappaB pathway andmutations in the valosin-containing protein (VCP) gene. This geneencodes VCP, which has a role in targeting the inhibitor of NFkappaB fordegradation by the proteasome (Daroszewska. A. and Ralston, S. H., Nat.Clin. Pract. Rheumatol. 2 (2006) 270-277). Targeted CSF-1R inhibitorsprovide an opportunity to block the deregulation of the RANKL signalingindirectly and add an additional treatment option to the currently usedbisphosphonates.

Cancer therapy induced bone loss especially in breast and prostatecancer patients is an additional indication where a targeted CSF-1Rinhibitor could prevent bone loss (Lester, J. E., et al., Br. J. Cancer94 (2006) 30-35). With the improved prognosis for early breast cancerthe long-term, consequences of the adjuvant therapies become moreimportant as some of the therapies including chemotherapy, irradiation,aromatase inhibitors and ovary ablation affect bone metabolism bydecreasing the bone mineral density, resulting in increased risk forosteoporosis and associated fractures (Lester, J. E., et al., Br. J.Cancer 94 (2006) 30-35). The equivalent to adjuvant aromatase inhibitortherapy in breast cancer is androgen ablation therapy in prostate cancerwhich leads to loss of bone mineral density and significantly increasesthe risk of osteoporosis-related fractures (Stoch, S. A., et al., J.Clin. Endocrinol. Metab. 86 (2001) 2787-2791).

Targeted inhibition of CSF-1R signaling is likely to be beneficial inother indications as well when targeted cell types include osteoclastsand macrophages e.g. treatment of specific complications in response tojoint replacement as a consequence of rheumatoid arthritis. Implantfailure due to periprosthetic bone loss and consequent loosing ofprostheses is a major complication of joint replacement and requiresrepeated surgery with high socioeconomic burdens for the individualpatient and the health-care system. To date, there is no approved drugtherapy to prevent or inhibit periprosthetic osteolysis (Drees, P., etal., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).

Glucocorticoid-induced osteoporosis (GIOP) is another indication inwhich a CSF-1R inhibitor could prevent bone loss after longtermglucocorticocosteroid use that is given as a result of variousconditions among those chronic obstructive pulmonary disease, asthma andrheumatoid arthritis (Guzman-Clark, J. R., et al., Arthritis Rheum. 57(2007) 140-146; Feldstein, A. C., et al., Osteoporos. Int. 16 (2005)2168-2174).

Rheumatoid arthritis, psioratic arthritis and inflammatory arthriditiesare in itself potential indications for CSF-1R signaling inhibitors inthat they consist of a macrophage component and to a varying degree bonedestruction (Ritchlin, C. T., et al., J. Clin. Invest. 111 (2003)821-831). Osteoarthritis and rheumatoid arthritis are inflammatoryautoimmune disease caused by the accumulation of macrophages in theconnective tissue and infiltration of macrophages into the synovialfluid, which is at least partially mediated by M-CSF. Campbell, I., K.,et al., J. Leukoc, Biol. 68 (2000) 144-150, demonstrated that M-CSF isproduced by human-joint tissue cells (chondrocytes, synovialfibroblasts) in vitro and is found in synovial fluid of patients withrheumatoid arthritis, suggesting that it contributes to the synovialtissue proliferation and macrophage infiltration which is associatedwith the pathogenesis of the disease. Inhibition of CSF-1R signaling islikely to control the number of macrophages in the joint and alleviatethe pain from the associated bone destruction. In order to minimizeadverse affects and to further understand the impact of the CSF-1Rsignaling in these indications, one method is to specifically inhibitCSF-1R without targeting a myriad other kinases, such as Raf kinase.

Recent literature reports correlate increased circulating M-CSF withpoor prognosis and atherosclerotic progression in chronic coronaryartery disease (Saitoh, T., et al., J. Am. Coll. Cardiol. 35 (2000)655-665; Ikonomidis, I., et al., Eur. Heart. J. 26 (2005) p. 1618-1624);M-CSF influences the atherosclerotic process by aiding the formation offoam cells (macrophages with ingested oxidized LDL) that express CSF-1Rand represent the initial plaque (Murayama, T., et al., Circulation 99(1999) 1740-1746).

Expression and signaling of M-CSF and CSF-1R is found in activatedmicroglia. Microglia, which are resident macrophages of the centralnervous system, can he activated by various insults, including infectionand traumatic injury, M-CSF is considered a key regulator ofinflammatory responses in the brain and M-CSF levels increase in HIV-1,encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis asa consequence of autocrine signaling by M-CSF/CSF-1R results ininduction of inflammatory cytokines and nitric oxides being released asdemonstrated by e.g. using an experimental neuronal damage model (Hao,A. J., et al., Neuroscience 112 (2002) 889-900; Murphy, G. M., Jr., etal., J. Biol. Chem. 273 (1998) 20967-20971). Microglia that haveincreased expression of CSF-1R are found to surround plaques in AD andin the amyloid precursor protein V717F transgenic mouse model of AD(Murphy, G. M, Jr., et al., Am. J. Pathol. 157 (2000) 895-904). On theother hand op/op mice with fewer microglia in the brain resulted infibrilar deposition of A-beta and neuronal loss compared to normalcontrol suggesting that microglia do have a neuroprotective function inthe development of AD lacking in the op/op mice (Kaku, M., et al., BrainRes. Brain Res. Protoc. 12 (2003) 104-108).

Expression and signaling of M-CSF and CSF-1R is associated withinflammatory bowel disease (IBD) (WO 2005/046657). The term“inflammatory bowel disease” refers to serious, chronic disorders of theintestinal tract characterized by chronic inflammation at various sitesin the gastrointestinal tract, and specifically includes ulcerativecolitis (UC) and Crohn's disease.

The invention comprises an antibody binding to human CSF-1R comprisingthe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor the treatment of cancer.

The invention comprises an antibody binding to human CSF-1R comprisingthe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor the treatment of bone loss.

The invention comprises an antibody binding to human CSF-1R comprisingthe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor the prevention or treatment of metastasis.

The invention comprises an antibody binding to human CSF-1R comprisingthe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor treatment of inflammatory diseases.

The invention comprises the use of an antibody comprising the antibodybinding to human CSF-1R, wherein the antibody comprises the abovementioned epitope binding properties or alternatively by the abovementioned amino acid sequences and amino acid sequence fragments for thetreatment of cancer or alternatively for the manufacture of a medicamentfor the treatment of cancer.

The invention comprises the use of an antibody comprising the antibodybinding to human CSF-1R, wherein the antibody comprises the abovementioned epitope binding properties or alternatively by the abovementioned amino acid sequences and amino acid sequence fragments for thetreatment of bone loss or alternatively for the manufacture of amedicament for the treatment of bone loss.

The invention comprises the use of an antibody comprising the antibodybinding to human CSF-1R, wherein the antibody comprises the abovementioned epitope binding properties or alternatively by the abovementioned amino acid sequences and amino acid sequence fragments for theprevention or treatment of metastasis or alternatively for themanufacture of a medicament for the prevention or treatment ofmetastasis.

The invention comprises the use of an antibody comprising the antibodybinding to human CSF-1R, wherein the antibody comprises the abovementioned epitope binding properties or alternatively by the abovementioned amino acid sequences and amino acid sequence fragments fortreatment of inflammatory diseases or alternatively for the manufactureof a medicament for the treatment of inflammatory diseases.

A further embodiment of the invention is a method for the production ofan antibody against CSF-1R wherein the sequence of a nucleic acidencoding the heavy chain of a human IgG1 class antibody binding to humanCSF-1R according to the invention said modified nucleic acid and thenucleic acid encoding the light chain of said antibody are inserted intoan expression vector, said vector is inserted in a eukaryotic host cell,the encoded protein is expressed and recovered from the host cell or thesupernatant.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COScells, yeast, or E. coli cells, and the antibody is recovered from thecells (from the supernatant or after cells lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified, or substantially pure form. Purification isperformed in order to eliminate other cellular components or othercontaminants, e.g. other cellular nucleic acids or proteins, by standardtechniques, including alkaline/SDS treatment, CsCl banding, columnchromatography, agarose gel electrophoresis, and others well known inthe art. See Ausubel, F., et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl, Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; Norderhaug, L., et al., J. Immunol. Methods 204(1997) 77-87. A preferred transient expression system (FMK 293) isdescribed by Schlaeger, E.-J. and Christensen. K., in Cytotechnology 30(1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

Nucleic acid molecules encoding amino acid sequence variants ofanti-CSF-1R antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of humanized anti-CSF-1Rantibody.

The heavy and light chain variable domains according to the inventionare combined with sequences of promoter, translation initiation,constant region, 3′ untranslated region, polyadenylation, andtranscription termination to form expression vector constructs. Theheavy and light chain expression constructs can be combined into asingle vector, co-transfected, serially transfected, or separatelytransfected into host cells which are then fused to form a single hostcell expressing both chains.

In another aspect, the present invention provides a composition, e.g. apharmaceutical composition, containing one or a combination ofmonoclonal. antibodies, or the antigen-binding portion thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption/resorption delaying agents, and the likethat are physiologically compatible. Preferably, the carrier is suitablefor injection or infusion.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the preparation of sterileinjectable solutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. In addition towater, the carrier can be, for example, an isotonic buffered salinesolution.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient (effectiveamount). The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The invention comprises the use of the antibodies according to theinvention for the treatment of a patient suffering from cancer,especially from colon, lung or pancreas cancer.

The invention comprises also a method for the treatment of a patientsuffering from such disease.

The invention further provides a method for the manufacture of apharmaceutical composition comprising an effective amount of an antibodyaccording to the invention together with a pharmaceutically acceptablecarrier and the use of the antibody according to the invention for sucha method.

The invention further provides the use of an antibody according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from cancer.

The invention also provides the use of an antibody according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from cancer.

EXAMPLES

The examples and figures are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1

Generation of a Hybridoma Cell Line Producing Anti-CSF-1R Antibodies

Immunization Procedure of NMRI Mice

NMRI mice were immunized with an expression vector pDisplay™(Invitrogen, USA) encoding the extracellular domain of huCSF-1R byutilizing electroporation. Every mouse was 4 times immunized with 100 μgDNA. When serum titers of anti-huCSF-1R were found to be sufficient,mice were additionally boosted once with 50 μg of a 1:1 mixture huCSF-1RECD/huCSF-1R ECDhuFc chimera in 200 μl PBS intravenously (i.v.) 4 and 3days before fusion.

Antigen Specific ELISA

Anti-CSF-1R titers in sera of immunized mice were determined by antigenspecific ELISA.

0.3 μg/ml huCSF-1R-huFc chimera (soluble extracellular domain) wascaptured on a streptavidin plate (MaxiSorb; MicroCoat, DE, Cat.No.11974998/MC1099) with 0.1 mg/ml biotinylated anti Fcγ (JacksonImmunoResearch., Cat.No. 109-066-098) and horse radish peroxidase(HRP)-conjugated F(ab′)₂ anti mouse IgG (GE Healthcare, UK,Cat.No.NA9310V) diluted 1/800 in PBS/005% Tween20/0.5% BSA was added.Sera from all taps were diluted 1/40 in PBS/0.05% Tween20/0.5% BSA andserially diluted up to 1/1638400. Diluted sera were added to the wells.Pre-tap serum was used as negative control. A dilution series of mouseanti-human CSF-1R Mab3291 (R&D Systems, UK) from 500 ng/ml to 0.25 ng/mlwas used as positive control. All components were incubated together for1.5 hours, Wells were washed 6 times with PBST (PBS/0.2% Tween20) andassays were developed with freshly prepared ABTS® solution (1 mg/ml)(ABTS: 2,2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) for 10minutes at RT. Absorbance was measured at 405 nm.

Hybridoma Generation

The mouse lymphocytes can be isolated and fused with a mouse myelomacell line using PEG based standard protocols to generate hybridomas. Theresulting hybridomas are then screened for the production ofantigen-specific antibodies. For example, single cell suspensions ofsplenic derived lymphocytes from immunized mice are fused to Ag8non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) with 50%PEG. Cells are plated at approximately 10⁴ in flat bottom 96 well microtiter plate, followed by about two weeks incubation in selective medium.Individual wells are then screened by ELISA for human anti-CSF-1Rmonoclonal 104 and IgG antibodies. Once extensive hybridoma growthoccurs, the antibody secreting hybridomas are replated, screened again,and if still positive for human IgG, anti-CSF-1R monoclonal antibodies,can he subcloned by FACS. The stable subclones are then cultured invitro to produce antibody in tissue culture medium for characterization.Antibodies according to the invention could be selected using thedetermination of the binding of anti-CSF-1R antibodies to human CSF-1Rfragment delD4 and to human CSF-1R Extracellular Domain (CSF-1R-ECD) asdescribed in Example 4, as well as the determination of growthinhibition of NIH3T3 cells transfected with wildtype CSF-1R (liganddependent signalling) or mutant CSF-1R L301S Y969F (ligand independentsignalling) under treatment with anti-CSF-1R monoclonal antibodies asdescribed in Example 5.

Culture of Hybridomas

Generated muMAb hybridomas were cultured in RPMI 1640 (PAN—Catalogue No.(Cat. No.) PO4-17500) supplemented with 2 mM L-glutamine (GIBCO—Cat. No.35050-038), 1 mM Na-Pyruvat (GIBCO—Cat. No. 11360-039), 1× NEAA(GIBCO—Cat. No. 11140-035), 10% FCS (PAA—Cat. NoA15-649), 1× Pen Strep(Roche Cat. No. 1074440), 1× Nutridoma CS (Roche—Cat. No.1363743), 50 μMMercaptoethanol (GIBCO—Cat. No.31350-010) and 50 U/ml IL 6 mouse(Roche—Cat. No. 1 444 581) at 37° C. and 5% CO₂. Some of the resultingmouse antibodies have been humanized (e.g. Mab 2F11) and been expressedrecombinantly.

Example 2

Inhibition of CSF-1 Binding to CSF-1R (ELISA)

By setting-up this assay to first allow for anti-CSF-1R antibody bindingto the CSF-1R-ECD followed by detection of ligand not bound to thereceptor both-ligand displacing antibodies and dimerization inhibitoranti-CSF-1R antibodies—can be tested. The test was performed on 384 wellmicrotiter plates (MicroCoat, DE, Cat.No. 464718) at RT. After eachincubation step plates were washed 3 times with PBST.

At the beginning, plates were coated with 0.5 mg/ml goat F(ab′)2biotinylated anti Fcγ (Jackson ImmunoResearch, Cat.No. 109-006-170) for1 hour (h).

Thereafter the wells were blocked with PBS supplemented with 0.2%Tween®-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 0.5 h. 75 ng/ml ofhuCSF-1R-huFc chimera (which forms the dimeric soluble extracellulardomain of huCSF-1R) was immobilized to plate for 1 h. Then dilutions ofpurified antibodies in PBS/0.05% Tween20/0.5% BSA were incubated for 1h. After adding a mixture of 3 ng/ml CSF-1 (Biomol, DE, Cat.No. 60530),50 ng/ml biotinylated anti CSF-1 clone BAF216 (R&D Systems,UK) and1:5000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat.No.11089153001) for 1 h the plates were washed 6 times with PBST. AntiCSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US), which inhibits theligand-receptor interaction, was used as positive control. Plates weredeveloped with freshly prepared BM blue® POD substrate solution (BMblue®: 3,3′-5,5′-Tetramethylbenzidine, Roche Diagnostics GmbH, DE,Cat.No. 11484281001) for 30 minutes at RT. Absorbance was measured at370 nm. A decrease of absorbance is found, if the anti-CSF-1R antibodycauses a release of CSF-1 from the dimeric complex. All anti-CSF-1Rantibodies showed significant inhibition of the CSF-1 interaction withCSF-1R (see Table 1). Anti CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, USsee also Sherr, C. J. et al., Blood 73 (1989) 1786-1793), which inhibitsthe ligand-receptor interaction, was used as reference control.

TABLE 1 Calculated IC50 values for the inhibition of the CSF-1/CSF-1Rinteraction IC50 CSF-1/CSF-1R CSF-1R Mab Inhibition [ng/ml] Mab 2F1119.3 Mab 2E10 20.6 Mab 2H7 18.2 Mab 1G10 11.8 SC-2-4A5 35.2

Example 3

Inhibition of CSF-1-Induced CSF-1R Phosphorylation in NIH3T3-CSF-1RRecombinant Cells

4.5×10³ NTH 3T3 cells, retrovirally infected with an expression vectorfor full-length CSF-1R, were cultured in DMEM (PAA Cat. No. E15-011), 2mM L-glutamine (Sigma, Cat.No.G7513, 2 mM Sodium pyruvate, 1×nonessential aminoacids, 10% FKS (PAA, Cat.NoA15-649) and 100 μg/mlPenStrep (Sigma, Cat.No. P4333 [10 mg/ml]) until they reachedconfluency. Thereafter cells were washed with serum-free DMEM media (PAACat.No.E15-011) supplemented with sodium selenite [5 ng/ml] (Sigma,Cat.No. S9133), transferrin [10 μg/ml] (Sigma, Cat.No. T8158), BSA [400μg/ml] (Roche Diagnostics GmbH, Cat.No. 10735078), 4 mM L-glutamine(Sigma, Cat.No. G7513), 2 mM sodium pyruvate (Gibco, Cat.No. 11360), 1×nonessential aminoacids (Gibco, Cat: 11140-035), 2-mercaptoethanol [0.05mM] (Merck, Cat.No. M7522), 100 μg/ml and PenStrep (Sigma, Cat. No.P4333) and incubated in 30 μl of the same medium for 16 hours to allowfor receptor up-regulation. 10 μl of diluted anti-CSR-1R antibodies wereadded to the cells for 1.5 h. Then cells were stimulated with 10 μl of100 ng/ml huM-CSF-1 (Biomol Cat.No. 60530) for 5 min. After theincubation, supernatant was removed, cells were washed twice with 80 μlof ice-cold PBS and 50 μl of freshly prepared ice-cold lysis buffer (150mM NaCl/20 mM Tris pH 7.5/1 mM EDTA/1 mM EGTA/1% Triton X-100/l proteaseinhibitor tablet (Roche Diagnostics GmbH Cat.No. 1 836 170) per 10 mlbuffer_(/)10 μl/ml phosphatase inhibitor cocktail 1 (Sigma Cat.No.P-2850, 100× Stock)_(/) 10 μl/ml protease inhibitor 1 (SigmaCat.No.P-5726, 1.00× Stock)/10 μl/ml 1 M NaF) was added. After 30minutes on ice the plates were shaken vigourously on a plateshaker for 3minutes and then centrifuged 10 minutes at 2200 rpm (Heraeus Megafuge10).

The presence of phosphorylated and total CSF-1 receptor in the celllysate was analyzed with Elisa. For detection of the phosphorylatedreceptor the kit from R&D Systems (Cat. No. DYC3268-2) was usedaccording to the instructions of the supplier. For detection of totalCSF-1R 10 μl of the lysate was immobilized on plate by use of thecapture antibody contained in the kit. Thereafter 1:750 dilutedbiotinylated anti CSF-1R antibody BAF329 (R&D Systems) and 1:1000diluted streptavidin-HRP conjugate was added. After 60 minutes plateswere developed with freshly prepared ABTS® solution and the absorbancewas detected. Data were calculated as % of positive control withoutantibody and the ratio value phosphoitotal receptor expressed. Thenegative control was defined without addition of M-CSF-1. Anti CSF-1R,SC 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C. J. et al.,Blood 73 (1989) 1786-1793), which inhibits the ligand-receptorinteraction, was used as reference control.

TABLE 2 Calculated IC50 values for the inhibition of CSF-1 receptorphosphorylation. IC50 CSF-1R Phosphorylation CSF-1R Mab [ng/ml] Mab 2F11219.4 Mab 2E10 752.0 Mab 2H7 703.4 Mab 1G10 56.6 SC-2-4A5 1006.6

Example 4

Determination of the Binding of Anti-CSF-1R Antibodies to Human CSF-1RFragment delD4 and to Human CSF-1R Extracellular Domain (CSF-1R-ECD)

Preparation of Human CSF-1R Extracellular Domain (CSF-1R-ECD)(Comprising the Extracellular Subdomains D1-D5, hCSF-1R-ECD) of SEQ IDNO: 64:

pCMV-preS-Fc-hCSF-1R-ECD (7836 bp) encodes the complete ECD of humanCSF-1R (SEQ ID NO: 64) C-terminally fused to a PreScission proteasecleavage site, followed by aa100-330 of human IgG1 and a 6× His-Tag,under the control of CMV promoter. The natural signal peptide has beenvaried by insertion of amino acids G and S after the first M, in orderto create a BamHI restriction site.

Preparation of Human CSF-1R Fragment delD4 (Comprising the ExtracellularSubdomains D1-D3 and D5,hCSF-1R-delD4) of SEQ ID NO: 65:

hCSF R-delD4-V1-PreSc-hFc-His was cloned from pCMV-preS-Fc-hCSF-1R-ECDby means of the Stratagene QuikChange XL site-directed mutagenesisprotocol, using delD4-for with sequenceCACCTCCATGTTCTTCCGGTACCCCCCAGA.GGTAAG (SEQ ID NO: 68) as the forwardprimer and delD4-rev with the reverse complement sequence as the reverseprimer. A protocol variation published in BioTechniques 26 (1999) 680was used to extend both primers in separate reactions in three cyclespreceeding the regular Stratagene protocol:

Two separate 50 μl reaction mixtures were set up according to themanufacturer's manual, each containing 10 ng plasmidpCMV-preS-Fc-hCSF1R-ECD as the template and 10 pM of one of the primersdelD4-for or delD4-rev, and 0.5 μl Pfu DNA polymerase as provided withthe kit. Three PCR cycles 95° C. 30 sec/55° C. 60 sec/68° C. 8 min wererun, then 25 μl each of both reaction mixtures were combined in a newtube and 0.5 μl fresh Pfu DNA polymerase were added. The regular PCRprotocol with 18 temperature cycles as specified by Stratagene in thekit manual was carried out, followed by 2 hrs final digestion with theDpn1 restriction enzyme provided with the kit. Clones bearing thedeletion were detected by digestion with Cel II and Not I and verifiedby sequencing.

Protein was prepared by transient transfection in the Hek293 FreeStylesuspension cell system (Invitrogen) according to the manufacturer'sspecifications. After 1 week 500 ml supernatant was filtered and loadedonto a 1 ml HiTrap MabSelect Xtra (GE healthcare) protein A column (0.2ml/min). The colomn was washed first with PBS, then with 50 mM Tris/150mM NaCl/1 mM EDTA/pH 7.3. 75 μl PreScission Protease (GE #27-0843-01)diluted in 375 μl of the same buffer were loaded onto the column and theclosed column was incubated over night at 4° C. with rolling. The columnwas mounted on top of a 1 ml GSTrap FF column (GE helthcare) and thedesired protein was eluted (0.2 ml/min, 0.2 ml fractions). Pooledfractions were concentrated from 1.8 ml to 0.4 ml by centrifugalultrafiltration via a 3 k Nanosep and chromatographed over an S200 HRSEC in PBS (0.5 ml/min).

Human CSF-1R fragment delD4 was obtained in two fractions as a dimericmolecule (pool1, V=1.5 ml; c=0.30 mg/ml; apparent mass on SDS page 83kDa, reduced 62 kDa) and as the monomer (pool 2, V=1.4 ml; c=0.25 mg/mlapparent mass on SDS page 62 kDa). The dimeric form was used for allexperiments.

Determination of the Binding of Anti-CSF-1R Antibodies to Human CSF-1RFragment delD4 and to Human CSF-1R Extracellular Domain (CSF-1R-ECD)(Binding Signals as Response Units (RU):

Instrument: Biacore T100 (GE Healthcare)

-   -   Software: T100 Control, Version 2.0.1        -   T100 Evaluation, Version 2.0.2

Assayformat Chip: CMS

Temperature: 25° C.

CSF-1R fragments were immobilized via amine coupling. To compare thebinding of different anti-CSF-1R antibodies according to the inventionone concentration of the test antibody was injected. Anti CSF-1R Mah3291(R&D-Systems) and SC 2-4A5 (Santa Cruz Biotechnology, US—see also Sherr,C. J. et al., Blood 73 (1989) 1786-1793), was used as reference control,anti-CCR5 m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18 Aug. 2004 atDSMZ) as negative control, all under the same conditions as theanti-CSF-1R antibodies according to the invention.

Amine Coupling of CSF-1R Fragments

Standard amine coupling according to the manufacturer's instructions:running buffer: PBS-T (Roche: 11 666 789+0.05% Tween20: 11 332 465),activation by mixture of EDC/NHS, injection of human CSF-1R fragmentdelD4 (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO:65) and human CSF-1R Extracellular Domain (CSF-1R-ECD) (comprising theextracellular subdomains D1-D5) (SEQ ID NO: 64) for 600 seconds at flowrate 10 μl/min; diluted in coupling buffer NaAc, pH 5.0, c=10 μg/mL;finally remaining activated carboxyl groups were blocked by injection of1 M Ethanolamin.

Binding of <CSF-1R>Mab 2F11, Mab 2E10, Mab 3291 and sc2-4A5 and OtherAnti-CW4R Antibodies to Human CSF-1R Fragment delD4 and Human CSF-1RExtracellular Domain (CSF-1R-ECD) at 25° C.

Running buffer: PBS-T (Roche: 11 666 789+0.05% Tween20: 11 332 465)

Analyte Sample:

Binding was measured at a flow rate of 30 μL/min by one injection of theanalyte with concentration c=10 nM. (for Mab 1G10, Mab 2H7 and humanizedhMab 2F11-e7 in second experiment) Each injection was 700 seconds long,followed by a dissociation phase of 180 seconds. Final regeneration wasperformed after each cycle using 50 mM NaOH, contact time 60 seconds,flow rate 30 μL/min.

Signals were measured by a report point 10 seconds after end ofinjection. Reference signals (signals from a blank reference flow cell(treated with EDC/NHS and ethanolamine, only) were subtracted to givethe binding signals (as RU). If binding signals of nonbinding antibodieswere slightly below 0 (Mab 2F11=−3; Mab 2E10=−2; Mab 1G10=−6, Mab2H7=−9; and humanized hMab 2F11-e7=−7) the values were set as 0.

TABLE 3a Binding of <CSF-1R> MAbs to human CSF-1R fragment delD4 andCSF-1R-ECD and ratio at 25° C., measured by SPR Ratio of binding Bindingof anti-CSF1R Binding to to CSF- antibodies to CSF1R delD4 1R-ECDfragment delD4/to [RU] [RU] CSF-1R-ECD Mab 3291 1015 627 1015/627 = 1.61sc2-4A5 374 249 374/249 = 1.50 Mab 2F11 0 176 0/176 = 0 hMab 2F11-e7 0237 0/237 = 0 Mab 2E10 0 120 0/120 = 0 Mab 1G10 0 2708 0/2708 = 0 Mab2H7 0 147 0/147 = 0 m<CCR5>Pz03.1C5 2 5 —

Mab 2F11 and Mab 2E10 showed binding to the human CSF-1R ExtracellularDomain (CSF-1R-ECD) (see FIG. 2b ); however no binding was detected toCSF-1R fragment delD4. (see FIG. 2a ).

Sc2-4A5 and MAB3291 showed binding to CSF-1R-ECD and to del D4 (seeFIGS. 2b and 2a ).

Thus the ratio of binding of anti-CSF-1R antibodies Mab 2F11 and Mab2E10 to CSF1R fragment delD4/to CSF-1R-ECD was clearly below 1:50(=0.02), while the binding ratio of MAB3291 and Sc2-4A5 were 1.61 and1.50, respectively and were highly above 1:50 (=0.02). Negative controlantibody m<CCR5>Pz03.1C5 did not show any binding (as expected).

Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 showed binding to the humanCSF-1R Extracellular Domain (CSF-1R-ECD) (see FIG. 2d ); however nobinding was detected to CSF-1R fragment delD4, (see FIG. 2c ). Thus theratio of binding of anti-CSF1R antibodies Mab 1G10, Mab 2H7 andhumanized hMab 2F11-e7 to CSF1R fragment delD4/to CSF-1R-ECD was clearlybelow 1:50 (=0.02).

In a further experiment anti-CSF-1R antibodies 1.2.SM (ligand displacingCSF-1R antibody described in WO2009026303), CXHG6 (ligand displacingCSF-1R antibody described in WO 2009/112245), the goat polyclonalanti-CSF-1R antibody ab10676 (abeam) were investigated. Anti-CSF-1Rantibody Mab3291 (R&D-Systems) was used as reference control. Anti-CCR5m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18 Aug. 2004 at DSMZ) wasused as negative control.

TABLE 3b Binding of <CSF-1R> MAbs to human CSF-1R fragment delD4 andCSF-1R-ECD and ratio at 25° C., measured by SPR Binding Ratio of bindingof Binding to to CSF- anti-CSF1R antibodies delD4 1R-ECD to CSF1Rfragment [RU] [RU] delD4/to CSF-1R-ECD MAB3291 1790 1222 1790/1222 =1.47 1.2.SM 469 704 469/704 = 0.67 CXIIG6 1983 1356 1983/1356 = 1.46ab10676 787 547 787/547 = 1.44 m<CCR5>Pz03.1C5 0 0 —

1.2.SM, CXIIG6, ab10676 and MAB3291 showed binding to CSF-1R-ECD and todel D4 (see FIGS. 2f and 2e ).

The binding ratio of 1.2.SM, CXHG6, ab10676 and MAB3291 was highly above1:50 (=0.02). Negative control antibody m<CCR5>Pz03.1C5 did not show anybinding (as expected).

Example 5

Growth Inhibition of NIH3T3-CSF-1R Recombinant Cells in 3D Culture UnderTreatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-assay)

NIH3T3 cells, retrovirally infected with either an expression vector forfull-length wildtype CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301S Y969F(SEQ ID NO: 63), were cultured in DMEM high glucose media (PAA,Pasching, Austria) supplemented with 2 mM L-glutamine, 2 mM sodiumpyruvate and non-essential amino acids and 10% fetal bovine serum(Sigma, Taufkirchen, Germany) on poly-HEMA(poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, Pa., USA))coated dishes to prevent adherence to the plastic surface. Cells areseeded in medium replacing serum with 5 ng/ml sodium selenite, 10 mg/mltransferrin, 400 μg/ml BSA and 0.05 mM 2-mercaptoethanol. When treatedwith 100 ng/ml huCSF-1 (Biomol, Hamburg, Germany) wtCSF-1R (expressingcells form dense spheroids that grow three dimensionally, a propertythat is called anchorage independence. These spheroids resemble closelythe three dimensional architecture and organization of solid tumors insitu. Mutant CSF-1R recombinant cells are able to form spheroidsindependent of the CSF-1 ligand. Spheroid cultures were incubated for 3days in the presence of different concentrations of antibody in order todetermine an IC50 (concentration with 50 percent inhibition of cellviability). The CellTiterGlo assay was used to detect cell viability bymeasuring the ATP-content of the cells.

TABLE 5a wtCSF-1R Mutant CSF-1R CSF-1R Mab IC₅₀ [μg/ml] IC₅₀ [μg/ml] Mab2F11 1.1 8.0 Mab 2E10 0.49 4.9 Mab 2H7 0.31 5.3 Mab 1G10 0.29 14.2 SC2-4A5 10.0 10.0

Reference control Mab R&D-Systems 3291 did not show inhibition of mutantCSF-1R recombinant cell proliferation.

In a further experiment the anti-CSF-1R antibody according to theinvention hMab 2F11-e7 and the anti-CSF-1R antibodies 1.2.SM (liganddisplacing CSF-1R antibody described in WO2009026303), CXIIG6 (liganddisplacing CSF-1R antibody described in WO 2009/112245), the goatpolyclonal anti-CSF-1R antibody ab10676 (abeam), and SC 2-4A5 (SantaCruz Biotechnology, US—see also Sherr, C. J. et al., Blood 73 (1989)1786-1793) were investigated.

Spheroid cultures were incubated for 3 days in the presence of differentconcentrations of antibody in order to determine an IC30 (concentrationwith 30 percent inhibition of cell viability). Maximum concentration was20 μg/ml The CellTiterGlo assay was used to detect cell viability bymeasuring the ATP-content of the cells.

TABLE 5b wtCSF-1R Mutant CSF-1R CSF-1R Mab IC₃₀ [μg/ml] IC₃₀ [μg/ml]hMab 2F11-e7  4.91 0.54 1.2.SM  1.19 >20 μg/ml (−19% inhibition at 20μg/ml = 19% stimulation) CXIIG6 >20 μg/ml (21% >20 μg/ml (−36%inhibition at 20 μg/ml) inhibition at 20 μg/ml = 36% stimulation)ab10676 14.15 >20 μg/ml (0% inhibition at 20 μg/ml) SC 2-4A5 16.62 2.56

Example 6

Growth Inhibition of BeWo Tumor Cells in 3D Culture Under Treatment withAnti-CSF-1R Monoclonal Antibodies (CellTiterGlo-assay)

BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K media(Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2 mML-glutamine. 5×10⁴ cells/well were seeded in 96-well poly-HEMA(poly(2-hydroxyethylmethacrylate)) coated plates containing F12K mediumsupplemented with 0.5% FBS and 5% BSA. Concomitantly, 200 ng/ml huCSF-1and 10 μg/ml of different anti-CSF-1R monoclonal antibodies were addedand incubated for 6 days. The CellTiterGlo assay was used to detect cellviability by measuring the ATP-content of the cells in relative lightunits (RLU). When BeWo spheroid cultures were treated with differentanti-CSF-1R antibodies (10 μg/ml) inhibition of CSF-1 induced growth wasobserved. To calculate antibody-mediated inhibition the mean RLU valueof unstimulated BeWo cells was subtracted from all samples. Mean RLUvalue of CSF-1 stimulated cells was set arbitrarily to 100%. Mean RLUvalues of cells stimulated with CSF-1 and treated with anti-CSF-1Rantibodies were calculated in % of CSF-1 stimulated RLUs. The Table 6shows the calculated data of growth inhibition of BeWo tumor cells in 3Dculture under treatment with anti-CSF-1R monoclonal antibodies; FIGS. 1aand b depicts normalized mean RLU values.

TABLE 6 % inhibition 10 μg/ml CSF-1R Mab antibody concentration CSF-1only 0 Mab 2F11 70 Mab 2E10 102 Mab 2H7 103 Mab 1G10 99 SC 2-4A5 39

Example 7

Inhibition of Human Macrophage Differentiation Under Treatment withAnti-CSF-1R Monoclonal Antibodies (CellTiterGlo-assay)

Human monocytes were isolated from peripheral blood using theRosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Tech.—Cat.No.15028). Enriched monocyte populations were seeded into 96 wellmicrotiterplates (2.5×10⁴ cells/well) in 100 μl RPMI 1640 (Gibco—Cat.No. 31870) supplemented with 10% FCS (GIBCO—Cat. No. 011-090014M), 4 mML-glutamine (GIBCO—Cat. No. 25030) and 1× PenStrep (Roche Cat. No. 1 074440) at 37° C. and 5% CO₂ in a humidified atmosphere. When 150 ng/mlhuCSF-1 was added to the medium, a clear differentiation into adherentmacrophages could be observed. This differentiation could he inhibitedby addition of anti-CSF-1R antibodies. Furthermore, the monocytesurvival is affected and could be analyzed by CellTiterGlo (CTG)analysis. From the concentration dependent inhibition of the survival ofmonocytes by antibody treatment, an IC₅₀ was calculated (see Table 7).

TABLE 7 CSF-1R Mab IC₅₀ [μg/ml] Mab 2F11 0.08 Mab 2E10 0.06 Mab 2H7 0.03Mab 1G10 0.06 SC 2-4A5 0.36

In a seperate test series humanized versions of Mab 2 F11, e.g. hMab2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, showed IC50 valuesof 0.07 μg/ml (hMab 2F11-c11), 0.07 μg/ml (hMab 2F11-d8), 0.04 μg/ml(hMab 2F11-e7) and 0.09 μg/ml (hMab 2F11-f12).

Example 8

Inhibition of Cynomolgous Macrophage Differentiation Under Treatmentwith Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-assay)

Cynomolgous monocytes were isolated from peripheral blood using the CD14MicroBeads non-human primate kit (Miltenyi Biotec—Cat.No. 130-091-097)according to the manufacturers description. Enriched monocytepopulations were seeded into 96 well microtiterplates (1-3×10⁴cells/well) in 100 μl RPMI 1640 (Gibco—Cat. No. 31870) supplemented with10% FCS (GIBCO—Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO—Cat. No.25030) and 1× PenStrep (Roche Cat. No. 1 074 440) at 37° C. and 5% CO₂in a humidified atmosphere. When 150 ng/ml huCSF-1 was added to themedium, a clear differentiation into adherent macrophages could beobserved. This differentiation could be inhibited by addition ofanti-CSF-1R antibodies. Furthermore, the monocyte survival, is affectedand could be analyzed by CellTiterGlo (CTG) analysis. The viability wasanalyzed at a concentration of 5 μg/ml antibody treatment (see Table 8).

TABLE 8 % inhibition (of survival) = CSF-1R Mab % survival (100% − %survival) Mab 2F11  4* 96 Mab 2E10  17** 83 Mab 2H7 8 92 Mab 1G10 2 98SC 2-4A5 31  69 *mean of four experiments (3 expts. using the murine, 1expt. using the chimeric mAb) **mean of two experiments using the murinemAb only

Example 9

Determination of the Binding Affinity of Anti-CSF-1R Antibodies to HumanCSF-1R

Instrument: BIACORE® A100

Chip: CMS (Biacore BR-1006-68)

Coupling: amine coupling

Buffer: PBS (Biacore BR-1006-72), pH 7.4, 35° C.

For affinity measurements 36 μg/ml anti mouse Fcγ antibodies (from goat,Jackson Immuno Reasearch JIR115-005-071) have been coupled to the chipsurface for capturing the antibodies against CSF-1R. Human CSF-1RExtracellular Domain (CSF-1R-ECD) (comprising the extracellularsubdomains D1-D5) (SEQ ID NO: 64) (R&D-Systems 329-MR or subclonedpCMV-presS-HisAvitag-hCSF-1R-ECD) was added in various concentrations insolution. Association was measured by an CSF-1R-injection of 1.5 minutesat 35° C.; dissociation was measured by washing the chip surface withbuffer for 10 minutes at 35° C. For calculation of kinetic parametersthe Langmuir 1:1 model was used.

TABLE 9 Affinity data measured by SPR CSF-1R Mab K_(D) (nM) k_(a) (1/Ms)k_(d) (1/s) t_(1/2) (min) Mab 2F11 0.29 1.77E⁺⁰⁵ 5.18E⁻⁰⁵ 223 Mab 2E100.2 1.52E⁺⁰⁵ 2.97E⁻⁰⁵ 389 Mab 2H7 0.21 1.47E⁺⁰⁵ 3.12E⁻⁰⁵ 370 Mab 1G100.36 1.75E⁺⁰⁵ 6.28E⁻⁰⁵ 184

In a separate biacore binding assay using the CSF-1R ECD (data notshown) some competition of the antibodies Mab 2E11 and Mab 2E10 with theantibody Ab SC-2-4A5 was shown. However Mab 2F11/Mab 2E10 do not bind tothe human CSF-1R fragment delD4, whereas Ab SC-2-4A5 binds to this delD4fragment (see Example 4 and FIG. 2a ). Thus the binding region of Mab2F11/Mab 2E10 is clearly distinct from the binding region of AbSC-2-4A5, but probably located in a vicinity area. In such competitionassay both antibodies Mab 2F11 and Mab 2E10 did not compete with Mab3291from R&D-Systems (data not shown).

Example 10

Determination of the Binding of Anti-CSF-1R Antibodies to Human CSF-1RFragment D1-D3

Instrument; Biacore T100 (GE Healthcare)

-   -   Software: T100 Control, Version 1.1.11.        -   B3000 Evaluation, Version 4.01        -   Scrubber, Version 2.0a

Assayformat Chip:CM5-Chip

Antibodies against CSF-1R were captured via amine coupled capturemolecules. Using the single cycle kinetics five increasingconcentrations of human CSF-1R fragment D1-D3 (SEQ ID NO: 66) wereinjected. Human CSF-1R fragment D1-D3 was subcloned intopCMV-presS-HisAvitag expression vector,

Anti CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US; Sherr, C. J., etal., Blood 73 (1989) 1786-1793) which inhibits the ligand-receptorinteraction, and Mab 3291 (R&D-Systems) were used as reference controls.

Capture molecules: Anti mouse Feγ antibodies (from goat, Jackson ImmunoReasearch JIR115-005-071) for antibodies according to the invention andthe R&D-Systems control Mab 3291 and Anti rat Fcγ antibodies (from goat,Jackson Immuno Reasearch JIR112-005-071) for the reference control antiCSF-1R SC 2-4A5.

Amine Coupling of Capture Molecules

Standard amine coupling according to the manufacturer's instructions:running buffer: HBS-N buffer, activation by mixture of EDC/NHS, aim forligand density of 2000 RU; the capture-Abs were diluted in couplingbuffer NaAc, pH 4.5, c=10 μg/mL; finally remaining activated carboxylgroups were blocked by injection of 1 M Ethanolamin.

Kinetic Characterization of Human CSF-1R Fragments D1-D3 Binding to MAbs<CSF-1R> at 37° C.

Running buffer: PBS (Biacore BR-1006-72)

Capturing of Mabs <CSF-1R> on flow cells 2 to 4: Flow 20 μL/min, contacttime 90 seconds, c(Abs<CSF-1R>)=50 nM, diluted with running buffer+1mg/mL BSA;

Analyte sample:

Single Cycle Kinetics was measured at a flow rate of 30 μL/min by fiveconsecutive injections of the analyte with concentrations, c =7.8,31.25, 125 500 and 2000 nM, without regeneration. Each injection was 30seconds long and followed by a dissociation phase of 120 Seconds for thefirst four injections, and finally 1200 seconds for the highestconcentration (=last injection).

Final regeneration was performed after each cycle using 10 mM Glycin pH1.5 (Biacore BR-1003-54), contact time 60 seconds, flow rate 30 μL/min.

Kinetic parameters were calculated by using the usual double referencing(control reference: binding of analyte to capture molecule; Flow Cell:subdomain CSF-1R concentration “0” as Blank) and calculation with model‘titration kinetics 1:1 binding with draft’.

TABLE 10 Affinity data for binding of human CSF-1R fragment D1-D3measured by SPR Sub CSF-1R Mab domain K_(D) (nM) k_(a) (1/Ms) k_(d)(1/s) t_(1/2) (min) Mab 2F11 D1-D3 no binding Mab 2E10 D1-D3 no bindingMab 2H7 D1-D3 not determined Mab 1G10 D1-D3 no binding SC-2-4A5 D1-D3 nobinding R&D-Systems D1-D3 5.4 2.2E⁺⁵ 1.2E⁻³ 9.6 3291

The antibodies Mab 2F11, Mab 2E10 and Mab 1G10 showed no binding tohuman CSF-1R fragment D1-D3

Also reference control-Ab SC-2-4A5 did not bind to human CSF-1R fragmentD1-D3.

The reference control Mab R&D-Systems 3291 showed binding to the humanCSF-1R fragment D1-D3.

Example 11

CSF-1 Level Increase During CSF-1R Inhibition in Cynomolgus Monkey

Serum CSF-1 levels provide a pharmacodynamic marker of CSF-1Rneutralizing activity of anti-human CSF-1R dimerization inhibitor hMab2F11-e7. One male and one female cynomolgus monkey per dosage group (1and 10 mg/kg) were intravenously administered anti-CSF1R antibody hMab2F11e7. Blood samples for analysis of CSF-1 levels were collected 1 weekbefore treatment (pre-dose), 2, 24, 48, 72, 96, 168 hours post-dose andweekly for two additional weeks. CSF-1 levels were determined using acommercially available ELISA kit (Quantikine® human M-CSF) according tothe manufacturer's instructions (R&D Systems, UK). Monkey CSF-1 levelwere determined by comparison with CSF-1 standard curve samples providedin the kit.

Administration of hMab 2F11-e7 induced a dramatic increase in CSF-1 by˜1000-fold, which depending on the dose administered lasted for 48 hr (1mg/kg) or 15 days (10 mg/kg). Hence, a dimerization inhibitor for CSF-1Roffers the advantage to not directly compete with the dramaticallyupregulated ligand for binding to the receptor in contrast to a liganddisplacing antibody.

Example 12

In Vivo Efficacy Tumor Growth Inhibition of Anti-CSF-1R Antibodies inBreast Cancer BT20 Xenograft Tumor Cells in SCID Beige Mice

The human breast cancer cell line BT-20 expresses human CSF-1R but lacksCSF-1 expression (Sapi, E. et al Cancer Res 59 (1999) 5578-5585). Sincethe mouse derived CSF-1 fails to activate human CSF-1R on the tumorcells recombinant human CSF-1 (Biomol, Hamburg, Germany) wassupplemented via osmotic minipumps (ALZET, Cupertino, Calif.) providinga continuous CSF-1 infusion rate of 2 μg/day (Martin, T. A.,Carcinogenesis 24 (2003) 1317-1323).

To directly compare the efficacy of an antibody interfering withdimerization of CSF-1R with a ligand displacing CSF-1R antibody wetested the chimeric anti-CSF-1R Mab 2F11 (antibody interfering withdimerization of CSF-1R) and 1.2.SM (ligand displacing CSF-1R antibodydescribed in WO2009026303) in the BT-20 xenograft model.

SCID beige mice (Charles River, Salzfeld, Germany) were subcutaneouslycoinjected with 1×107 cells BT-20 cells (ATCC HTB-19) and 100 μl ofMatrigel. Treatment of animals started at day of randomization at a meantumor volume of 100 mm3. Mice are treated once weekly i.p. with therespective antibodies (see FIG. 4) in 20 mM Histidine, 140 mM NaCl pH6.0 buffer. The tumor dimensions are measured by caliper beginning onthe staging day and subsequently 2 times per week during the wholetreatment period. Tumor volume is calculated according to NCI protocol(Tumor weight=1/2ab2, where “a” and “b” are the long and the shortdiameters of the tumor, respectively).

Tumor growth analysis is shown in FIG. 4. Inhibition of human CSF-1R ontumor cells with the chimeric anti-CSF-1R Mab 2F11 was statisticallymore efficacious in mediating tumor growth inhibition than anti-CSF-1Rantibody 1.2.SM (CSF-1R antibody described in WO2009026303).

Although the foregoing, invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not he construed aslimiting the scope of the invention. The disclosures of all patents,patent applications, scientific references, and Genbank Accession Nos.cited herein are expressly incorporated by reference in their entiretyfor all puroposes as if each patent, patent application, scientificreference, and Genbank Accesion No. were specifically and individuallyincorporated by reference.

The invention claimed is:
 1. An isolated antibody binding to humanCSF-1R, wherein the antibody comprises a heavy chain variable domain anda light chain variable domain, and wherein the heavy chain variabledomain comprises a CDR3 region comprising SEQ ID NO: 69, a CDR2 regioncomprising SEQ ID NO: 70, and a CDR1 region comprising SEQ ID NO: 71,and the light chain variable domain comprises a CDR3 region comprisingSEQ ID NO:72, a CDR2 region comprising SEQ ID NO: 73, and a CDR1 regioncomprising SEQ ID NO:
 74. 2. A pharmaceutical composition comprising anantibody according to claim
 1. 3. The pharmaceutical compositionaccording to claim 2, wherein said antibody is formulated in apharmaceutically acceptable carrier.
 4. The antibody according to claim1, wherein the antibody is human IgG1 subclass or is of human IgG4subclass.
 5. The antibody according to claim 1, wherein the antibody isa chimeric, single chain, multispecific, or humanized antibody.
 6. Apharmaceutical composition comprising an antibody according to claim 5.7. The antibody according to claim 1, wherein the antibody is an antigenbinding fragment.
 8. A pharmaceutical composition comprising an antibodyaccording to claim
 7. 9. The antibody according to claim 1, wherein theantibody is a monoclonal antibody.
 10. A pharmaceutical compositioncomprising an antibody according to claim
 9. 11. An isolated antibodybinding to human CSF-I R, wherein the antibody comprises a heavy chainvariable domain and a light chain variable domain, and wherein the heavychain variable domain comprises SEQ ID NO: 75 and the light chainvariable domain comprises SEQ ID NO:
 76. 12. A pharmaceuticalcomposition comprising an antibody according to claim
 11. 13. Thepharmaceutical composition according to claim 12, wherein said antibodyis formulated in a pharmaceutically acceptable carrier.
 14. The antibodyaccording to claim 11, wherein the antibody is human IgG1 subclass or isof human IgG4 subclass.
 15. The antibody according to claim 11, whereinthe antibody is a chimeric, single chain, multispecific, or humanizedantibody.
 16. A pharmaceutical composition comprising an antibodyaccording to claim
 15. 17. The antibody according to claim 11, whereinthe antibody is an antigen binding fragment.
 18. A pharmaceuticalcomposition comprising an antibody according to claim
 17. 19. Theantibody according to claim 11, wherein the antibody is a monoclonalantibody.
 20. A pharmaceutical composition comprising an antibodyaccording to claim 19.